Inhibitors of monocarboxylate transport

ABSTRACT

Methods of screening compounds for their ability to inhibit monocarboxylate transport, and methods of making and using such inhibitors to treat disorders associated with cellular proliferation, e.g., immune-mediated disorders and cancer.

[0001] This patent application claims the benefit of U.S. ProvisionalApplication Serial No. 60/329,318, filed 16 Oct. 2001, the specificationof which is hereby incorporated by reference.

[0002] This invention relates, inter alia, to methods of screeningcompounds for their ability to reduce monocarboxylate transport, andmethods of using such compounds to treat certain cancers andimmune-mediated disorders.

BACKGROUND OF THE INVENTION

[0003] The immune system has evolved to detect the presence of foreignorganisms such as bacteria, viruses and other pathogens, and to mountprotective immune responses to eliminate them. Under certaincircumstances, the induction of an immune response against foreignorganisms or tissues proves more harmful to the host than ignoring them.For example, asthma and allergies to food and extrinsic antigens such aspollen are believed to reflect inappropriate hypersensitivity responsesto otherwise harmless substances. In addition, transplant patients oftenexhibit strong immune responses against transplanted tissues. These aredetrimental to the survival of the transplanted organ and must belimited by administration of potent immunosuppressive drugs.

[0004] Under normal circumstances the immune system does not produceimmune responses to self-tissues and self-antigens. However, under someconditions immune responses are mounted against self-tissues in such anaggressive manner that they lead to destructive autoimmune diseases: forexample, rheumatoid arthritis, multiple sclerosis and type I diabetes.In these situations it would be desirable to reset the immune system sothat responses to self-antigens are silenced but without affectingprotective host defence mechanisms directed against exogenous antigens.

[0005] Most immune responses are initiated and controlled by helper Tlymphocytes, which respond to antigenic peptide fragments presented inassociation with MHC Class II molecules, and cytotoxic T lymphocytes,which respond to peptides presented in association with MHC Class Imolecules on specialised antigen presenting cells such as dendriticcells.

[0006] Full T cell activation requires two distinct signals from theantigen presenting cell. Signal 1 is antigen specific and is provided bythe interaction of the T cell receptor (TCR) with the MHC-peptidecomplex displayed by the antigen-presenting cell. Signal 2 is antigenindependent and involves the interaction of the co-stimulatory T cellmolecule, CD28, with its ligand, B7, on antigen presenting cells. Thesecell-surface interactions trigger downstream biochemical signallingpathways that ultimately result in IL2 transcription and T-cellactivation.

[0007] As a means of treating these immune-mediated disorders, researchprograms to date have typically concentrated on identifying compoundscapable of blocking IL2 transcription. The promoter region of the IL2gene includes a binding site for the Nuclear Factor of Activated T-cells(NFAT) transcription factor complex. This complex is composed of nuclearcomponents fos and jun and a cytoplasmic component NFAT_(c) thattranslocates to the nucleus after dephosphorylation by the phosphatasecalcineurin. The immunosuppressive macrolides cyclosporin A (CsA) andFK506 block transcription of the IL2 gene in T-lymphocytes by preventingformation of the NFAT complex (Crabtree, Cell 96:611-614, 1999).Complexes formed by the binding of CsA and FK506 to their respectiveimmunophilins, cyclophilin and FKBP12, inhibit calcineurin activity andthus block NFAT_(c) translocation. Although CsA and FK506 are potentimmunosuppressive drugs used clinically for the prevention of graftrejection, their long-term utility for the treatment of auto-immunedisease is limited by their side effect profile, includingnephrotoxicity. These adverse reactions appear to be related toinhibition of calcineurin activity, as the enzyme is expressed widelyacross mammalian tissues and has multiple functions.

[0008] A number of additional immunosuppressive therapies have now beendeveloped (Dumont, Opin. Ther. Patents 11:377-404, 2001). These includerapamycin, which disrupts the cytokine (e.g., IL2)-driven proliferationof T-cells, by interfering with the function of TOR (Target OfRapamycin), a kinase involved in the cytokine signalling pathway (Dumontand Su, Life Sci. 58:373-395, 1996). However, rapamycin has been shownto cause significant side effects, including thrombocytopenia andhyperlipidemia (Hong and Kahan, Semin. Nephrol. 20 (2):108-125, 2000).Antimetabolite approaches are also of utility for immunosuppression, asT-lymphocytes have been shown to be dependent on de novo synthesis ofribonucleotides (Fairbanks et al., J. Biol. Chem. 270(50):29682-29689,1995). Mycophenolate mofetil (MMF), which inhibits inosine monophosphatedehydrogenase (IMPDH), the regulatory enzyme of guanine nucleotidebiosynthesis (Allison and Eugui, Immunopharmacology 47:85-118, 2000), iseffective in reducing T-cell proliferation and has been used for thetreatment of graft rejection. However, MMF is rapidly glucoronidated invivo and is associated with gastrointestinal toxicity (Dumont, Curr.Opin. Invest. Drugs. 2 (3):357-363, 2001).

[0009] Screening programmes investigating NFAT-mediated transcriptiondirectly have been used to identify small molecule inhibitors of IL2production without the side effect profile of calcineurin inhibitors.Michne et al. discovered a class of quinazolinedione compounds that wereidentified by inhibition of NFAT-mediated gene transciption in a Jurkathuman leukemic T-cell line (Michne et al., J. Med. Chem. 38:2557-2569,1995). An example of the quinazolinedione class of compounds, WIN 61058,that inhibited NFAT-mediated transcription with a potency of 2 μM wasshown to block IL2 production in Jurkat T-cells and to inhibit a humanMixed Lymphocyte Reaction (MLR) (Baine et al., J. Immunol.154:3667-3677, 1995). A chemical programme based on WIN 61058 resultedin the identification of pyrrolopyrimidinedione inhibitors ofNFAT-mediated transcription with potencies as high as 2 nM (Michne etal., supra). We have confirmed that examples of thesepyrrolopyrimidinediones inhibit NFAT-mediated gene transcription inJurkat T-cells, but found that early IL2 production in response tomitogenic stimulation of peripheral blood mononuclear cells (PBMC) isnot significantly inhibited. The applicant's own chemical programme hasexemplified pyrimidinedione inhibitors that potently inhibit the humanMLR in the absence of inhibition of IL2 transcription. Contrary to themechanism proposed by Michne et al. (supra), the inventors have, for thefirst time, identified the mechanism of action of these pyrimidinedionecompounds as being blockade of monocarboxylate transport through themonocarboxylate transporter MCT1. This pioneering invention explains atotally new mechanism of action not foreshadowed in any way by the artin this highly competitive field, opening up new methods of treatment byinhibiting this mechanism of action and also new targets for identifyingimmunosuppressive agents.

[0010] WO 98/46606 (AstraZeneca), incorporated herein by reference,discloses a family of pyrazolo[3,4-d]pyrimidinedione compounds; WO98/54190 (AstraZeneca), incorporated herein by reference, discloses afamily of thieno[2,3-d]pyrimidinedione compounds; WO98/28301(AstraZeneca), incorporated herein by reference, discloses afamily of 5-substituted pyrrolo[3,4-d]pyrimidine-2,4-dione compounds; WO99/29695 (AstraZeneca), incorporated herein by reference, disclosescertain pyrrolo-, thieno-, furano-and pyrazolo-[3,4-d]pyridazinonecompounds; WO 00/12514 (AstraZeneca), WO 01/83489 (AstraZeneca),PCT/GB02/03399 (AstraZeneca), PCT/GB02/03250(AstraZeneca) andGB-A-2363377 (AstraZeneca), each incorporated herein by reference, eachdisclose certain thieno[2,3-d]pyrimidinedione compounds. Each of thesecompounds, which exhibit pharmacological activity, in particularimmunosuppressive activity, is disclosed for the first time herein toeffect this via inhibition of monocarboxylate transport.

[0011] MCT1 is a member of a family of monocarboxylate transporters thatmediate the influx and efflux of monocarboxylates, such as lactate andpyruvate, across cell membranes. The MCT proteins transportmonocarboxylates by a facilitative diffusion mechanism that requires theco-transport of protons (Poole and Halestrap, Am. J.Physiol.264:C761-C782, 1993; Halestrap and Price, Biochem. J 343:281-299, 1999).Nonselective small molecule inhibitors of the transporter have beenidentified, including 4,4′-di-isothiocyanatostilbene-2,2′-disulphonate(DTDS), α-cyano-4-hydroxycinnamate (CHC) and phloretin. These arenon-selective inhibitors with potencies in the μM range (IC50 values atrat MCT1: DIDS=50 μM; CHC=27 μM; phloretin=1 μM)(Poole and Halestrap,supra). The MCT1 protein, a 55 kDa protein, has been enriched from ratred blood cells (Poole et al., Biochem. J 320:817-824, 1996).Hydrophobicity analysis and studies on the membrane topology of rat MCT1have suggested a structure with 12 transmembrane segments andintracellular N- and C-terminal regions (Poole et al., supra).

[0012] The nomenclature for the MCT family is taken from Price et al.,Biochem. J 329 (2):321-328, 1998; and Halestrap and Price, Biochem. J343:281-299, 1999.

[0013] Roth et al. showed that addition of exogenous lactate to T-cellsresulted in inhibition of DNA synthesis and proliferation (Roth et al.,Cell. Immunol. 136:95-104, 1991) but that IL2 production was augmented(Droge et al., Cell. Immunol. 108:405-416, 1987). Studies on themetabolism of activated T-lymphocytes (thymocytes) have demonstratedthat the cells derive 86% of their energy supply by aerobic glycolysis,i.e., glycolytic breakdown of glucose to lactate (Brand and Hermfisse,FASEB J 11:388-39, 1997; Guppy et al., Eur. J Biochem. 212:95-99, 1993).The monocarboxylate transporter MCT4 has been characterised as atransporter with low affinity for lactate and pyruvate (Dimmer et al.,Biochem. J 350:219-227, 2000). Therefore, it has been suggested thatMCT4 is adapted to the release of lactate from glycolytically-activecells, whereas the high affinity transporter MCT1 transports lactaterequired for energy production into cells (Manning Fox et al.,J.Physiol. 529:285-293, 2000).

[0014] Zhao et al. (Diabetes 50:361-366, 2001) propose that, in someforms of Type II diabetes, MCT overexpression in the pancreatic isletcells could contribute to aberrant secretion of insulin, and, thereforepostulate that inhibitors of islet cell lactate transport, or of MCT1gene expression, could provide a therapeutic target for this disease.

[0015] Froberg et al. (Neuroreport 12(4):761-765, 2001) reportedincreased MCT1 expression in high grade glial neoplasms and speculatedthat it may provide a potential therapeutic target for treatment of someCNS neoplasms.

[0016] The monocarboxalate transporters MCT1 to MCT4 are known totransport monocarboxylate (Halestrap and Price, Biochem. J 343:281-299,1999).

SUMMARY OF THE INVENTION

[0017] It has now been discovered that compounds capable of blockingcellular monocarboxylate transport can inhibit cellular proliferation,and are therefore useful for treatment of various disorders associatedwith unwanted cellular proliferation. Accordingly, in a first aspect ofthe invention there is provided a method for identifying compound(s)that may have therapeutic potential, the method comprising determiningwhether a test compound decreases monocarboxylate transport activity,and, if the compound decreases such activity, identifying the compoundas having therapeutic potential. The term “decreasing monocarboxylatetransport activity” is intended to cover decreasing such activity by anymeans that specifically affects monocaraboxylate transport, e.g., byinhibiting the activity of an existing monocarboxylate transporterprotein molecule or by reducing the amount of cellular monocarboxylatetransporter protein present in a cell. Thus, the determining step mightinvolve, for example, determining whether the test compound directlyinhibits the activity of a monocarboxylate transport protein, ordetermining whether the compound reduces the level of expression or thetotal amount of a monocarboxylate transport protein in a cell.Particularly where inhibition of the protein's activity is the focus ofthe screening method, the protein would preferably be in a cell, a cellghost, a cell membrane fraction, or a lipid vesicle.

[0018] Methods of carrying out the determining step can include thesub-steps of (i) providing a cell expressing a monocarboxylatetransporter protein; (ii) contacting the cell with the test compound;and (iii) determining whether the test compound affects one or more ofthe following: monocarboxylate accumulation within the cell,monocarboxylate efflux from the cell, H+ efflux from the cell, or H+accumulation within the cell, as an indication that the test compoundinhibits the protein's monocarboxylate transport activity. Thedetermining step optionally employs an assay selected from the groupconsisting of rapid filtration of equilibrium binding mixtures,radioimmunoassays (RIA), fluorescence resonance energy transfer assays(FRET), scintillation proximity assay (SPA), measurement ofintracellular pH, and the use of labelled substrates to measuretransport.

[0019] In this and each of the other screening methods of the invention,the preferred target transport protein is MCT1, 2, 3, or 4, preferablyof a warm-blooded animal, and more preferably of a mammal such as ahuman, mouse, rat, guinea pig, hamster, rabbit, dog, cat, cow, horse,goat, sheep, pig, or non-human primate. The test compound can be anytype of compound, including in particular small molecules as well asproteins such as antibodies or antibody fragments.

[0020] In preferred embodiment of each of the screening methodsdescribed herein, the method is useful in identifying agents(s) that mayhave potential in treating a disorder associated with unwanted cellularproliferation, such as an immune-mediated disorder (e.g., transplantrejection and inflammation), cancer (e.g., non-central nervous system(CNS) or non-glial cell cancers), or blood vessel blockage, as inrestenosis. Accordingly, any of the screening methods can include anadditional step of further testing a compound that was identified in thescreen in a cellular proliferation assay. Such an assay could test, forexample, whether the compound inhibitis proliferation of cells such asactivated T lymphocytes, cancer cells in vitro, or cancer cells in vivo.Alternatively, the compound could be further tested in an in vivo or invitro model of inflammation, autoimmune disease, or transplantrejection.

[0021] In another method of the invention, there is provided a methodfor identifying a compound having therapeutic potential, the methodincluding the steps of (a) determining whether a test compound binds toa monocarboxylate transport protein; and (b) if the compound binds tothe protein, identifying the compound as having therapeutic potential.The determining step can include ascertaining the binding affinity ofthe compound for the protein, optionally in accordance with thetechniques described below. Alternatively or in addition, thedetermining step can include a competitive binding assay, using ascompetitive reagent a labelled second compound that specifically bindsto the protein. In a preferred embodiment, the determining stepcomprises providing a cell expressing the protein, or a cell membranepreparation derived from the cell, and contacting the test compound withthe cell or the preparation. The cell may naturally express the protein,or the protein may be a recombinant protein expressed by a cell that istransfected with a nucleic acid encoding the protein. In the lattercase, the cell prior to transfection would preferably express little orno monocarboxylate transport protein of the type being studied.

[0022] According to a further aspect of the invention, there is provideda method for determining whether a compound not known to be capable ofspecifically binding to a monocarboxylate transporter can specificallybind to a monocarboxylate transporter. This method comprises contactinga monocarboxylate transporter protein with the compound under conditionssuitable for binding, and detecting specific binding of the compound tothe transporter. Such a method is particularly applicable foridentifying potentially useful therapeutic compounds. In one embodimentthe transporter is presented within a natural or synthetic membrane. Forexample, the transporter could be presented within lipid vesicles, asdescribed by Lynch and McGiven (Biochem. J 244:503-508, 1987).

[0023] According to a further aspect of the invention, there is providedan assay for identifying compounds that inhibit monocarboxylatetransport in a cell, the assay comprising:

[0024] (a) contacting a cell or cell lysate comprising a monocarboxylatetransport polypeptide (or a DNA or RNA encoding the polypeptide) with atest compound; and

[0025] (b) detecting one or more of the following characteristics:

[0026] (i) the ability of the test compound to inhibit the ability ofthe monocarboxylate transport polypeptide to transport monocarboxylate,

[0027] (ii) the ability of the test compound to bind to themonocarboxylate transport polypeptide, and

[0028] (iii) the ability of the test compound to block expression of themonocarboxylate transport polypeptide.

[0029] According to a further aspect of the invention there is provideda method for identifying whether or not a compound may have potential intreating an immune-mediated disorder or cancer (particularly a cancerother than a CNS or glial cell cancer), which comprises contacting cellsexpressing a monocarboxylate transporter, or cell membrane preparationsthereof, with a compound not known to be capable of inhibitingmonocarboxylate transport, under conditions suitable for binding, anddetermining monocarboxylate transport activity, wherein the ability ofthe compound to inhibit monocarboxylate transport identifies thatcompound as having potential in treating an immune-mediated disorder orcancer.

[0030] According to a further aspect of the invention there is provideda method for determining whether a compound not known to be capable ofblocking monocarboxylate transport can block monocarboxylate transport,which comprises contacting cells expressing a monocarboxylatetransporter, or cell membrane preparations thereof, with the compoundunder conditions suitable for binding, and determining monocarboxylatetransport activity. This method can be employed, for example, todetermine the suitability of a compound for assessment as a potentialtherapeutic agent. Potential test therapeutic agents would possess IC50values of at least 10 μM, preferably at least 1 μM, for inhibition ofmonocarboxylate transport (IC50 being the concentration of compoundresulting in 50% inhibition of the response). In one embodiment themonocarboxylate transporter is expressed from nucleic acid exogenouslyintroduced into a cell. In another embodiment monocarboxylate transportis blocked as a result of the compound's specifically binding to themonocarboxylate transporter. In another embodiment monocarboxylatetransport is blocked as a result of the compound's impeding expressionof the monocarboxylate transporter. In a further embodiment the methodincludes a step of determining whether or not the compound is capable ofspecifically blocking monocarboxylate transport.

[0031] A compound is typically identified as an MCT inhibitor if itexhibits an inhibition constant, Ki, of less than or equal to 10 μM. Theinhibition constant, Ki, is the concentration of competing ligand thatwould occupy 50% of the binding sites if no radioligand were present inthe competitive binding assay. The Ki is calculated from the IC50 usingthe Cheng-Prusoff equation. IC50 values are determined as theconcentration of inhibitor that would displace 50% of radioligand A or C(described in Example 1 herein) as measured in filter binding assaysand/or the scintillation proximity assay(s) described herein.

[0032] According to a further aspect of the invention there is provideda method for identifying a compound that may have potential in treatingan immune-mediated disorder or cancer, comprising determining whetherthe compound is capable of inhibiting monocarboxylate transport activityof a cell.

[0033] Thus, in a further aspect of the invention there is provided useof a human monocarboxylate transporter, or a cell or cell membranepreparation comprising a monocarboxylate transporter protein, preferablyone selected from the group consisting of MCT1 through MCT4, in the invitro screening of compounds for their ability to treat animmune-mediated disorder or cancer, particularly non-CNS or non-glialcell cancer.

[0034] The term “monocarboxylate transport activity,” as used herein,refers to the ability of the transporter protein to facilitate transportof monocarboxylate molecules, such as lactate and pyruvate, across acell membrane. Such activity can be determined using various techniquesknown to the person skilled in the art, some of which are specificallydescribed herein in the context of detecting inhibition of suchactivity. According to a further aspect of the invention, there isprovided a compound, or a pharmaceutically acceptable salt thereof,identified by any of the screening methods of the invention. In oneembodiment, the compound will be capable of specifically inhibitingmonocarboxylate transport; preferably, the compound would be at leastten times as active (and preferably at least 50 or 100 times as active)against one of the four types of MCT proteins (MCT1, 2, 3, and 4) asagainst any other of the four. In another preferred embodiment, thecompound does not fall within any of Formulae I-IX as defined below. Inaddition, the compound preferably is not4,4′-di-isothiocyanatostilbene-2,2′-disulphonate (DIDS),α-cyano-4-hydroxycinnamate (CHC), or phloretin. In another embodiment,the compound is not a quinazolinedione, pyrimidinedione, or pyridazinonecompound. The compound is preferably not one specifically disclosed, bychemical name or by generic or specific formula, in any of the prior artreferenced herein.

[0035] According to a further aspect of the invention, there is provideda method of producing a pharmaceutical composition, which methodcomprises determining whether or not a compound is an MCT inhibitorusing any of the screening methods of the invention and furthermoremixing the compound identified therein, or a pharmaceutically activederivative thereof, with a pharmaceutically acceptable carrier.Alternatively, the method can comprise providing a compound that wasidentified as an MCT inhibitor using a screening assay of the invention,and mixing the compound with a pharmaceutically acceptable carrier ordiluent. In another aspect, the process includes the steps of carryingout one of the screening methods disclosed herein to identify a compoundwith therapeutic potential, and manufacturing a therapeutic compositioncomprising the compound in accordance with practices that ensure thesterility of the composition (e.g., Good Manufacturing Practicesespoused by regulatory agencies). The resulting composition can then belabelled for use in a method of treating a specified cell proliferativedisorder, such as an immune-mediated condition or cancer. Generally,such a label would describe the disorder and how the composition shouldbe administered to a patient in need thereof, including dosage.

[0036] In the context of this aspect of the invention, a derivative of acompound identified in a screen of the invention is a compound that hasbeen designed, synthesised and tested for MCT inhibitor activity basedon the structure of the parent compound initially identified in thescreen. Such a derivative compound is generally identified usingconventional structure activity relationship (SAR) studies. Furthermore,such derivative compounds will generally share significant structuralfeatures with the parent compound, but with one or more structuralmoieties altered. A derivative compound is likely to be one whosestructure has been optimised to make the compound more suitable fortherapeutic treatments, such as by removal of groups known to beassociated with toxic effects; being more bioavailable; having a longerhalf-life in vivo, etc.

[0037] According to a further aspect of the invention there is provideda method of producing a pharmaceutical composition that comprisesdetermining whether or not a compound is an MCT inhibitor using any ofthe screening methods of the invention; preparing derivative compoundsof this “parent” compound; testing these derivative compounds in one ofthe screening methods of the invention to identify a more activecompound; and mixing said more active compound identified therein with apharmaceutically acceptable carrier.

[0038] The components of any of the screening methods of the inventioncan be combined in a suitable kit of parts format. Thus, according to afurther aspect of the invention there is provided a kit for use in amethod for screening compounds for binding to or inhibition of a MCT,the kit comprising:

[0039] (i) a vessel containing a cell capable of expressing amonocarboxylate transporter protein or a cell membrane preparationcontaining a monocarboxylate transport protein; and

[0040] (ii) a vessel containing a labelled compound that specificallybinds to the monocarboxylate transporter protein.

[0041] The kit optionally includes instructions for use in a screeningassay of the invention, and optionally describes the utility of theassay in identifying compounds that are useful for treating cancerand/or immune-mediated diseases.

[0042] The invention includes a method of treating an animal (includinga human) subject in need of treatment for a disease or conditioncharacterized by T-cell activation or cellular proliferation (e.g., animmune-mediated disorder or cancer), the method comprising administeringto the subject a compound that inhibits cellular monocarboxylatetransporter activity. In this and each of the other methods of treatmentdescribed herein, the compound is preferably not a compound of formulaeI to IX. Alternatively, the preferred compound can be characterized asbeing other than a compound disclosed in any of InternationalPublication Nos. WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO00/12514, and WO 01/83489; International Application numbersPCT/GB02/03399 and PCT/GB02/03250; and UK Patent application numberGB-A-2363377. Furthermore, the compound is preferably not one disclosedin Michne et al., J. Med. Chem. 38:2557-2569 (1995); or Baine et al., J.Immunol. 154:3667-3677 (1995). It also is preferably not aquinazolinedione compound.

[0043] In another aspect, the method of treating a subject in need oftreatment for an immune-mediated disorder or cancer comprisesidentifying a subject as being in need of such treatment andadministering to the subject a compound that inhibits or otherwisedecreases the activity of a monocarboxylate transporter other than MCT1or MCT2. The transporter can be, for example, MCT3 or MCT4.

[0044] Also within the invention is a method of treating a patientsuffering from or likely to suffer from an immune-mediated disorder orcancer, the method comprising (i) identifying a compound as being aninhibitor of monocarboxylate transport in a cell, and (ii) administeringto the patient an effective amount of the compound. In this as well asthe other treatment methods disclosed herein, the compound can be abroad spectrum inhibitor capable of potently inhibiting at least twomonocarboxylate transport proteins. In another embodiment, the compoundis at least ten times as active against one of MCT1, 2, 3, and 4, asagainst any other of the four.

[0045] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer by a methodcomprising administration, to a human subject in need of treatment, of acompound that inhibits or otherwise decreases the activity of at leastone monocarboxylate transporter selected from the group consisting ofMCT3 and MCT4.

[0046] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer, comprisingadministering an effective amount of a pharmaceutical compositioncomprising an MCT inhibitor identifiable or identified by a screeningassay method of the invention to a subject in need thereof. In oneembodiment, the MCT inhibitor is a selective inhibitor.

[0047] Also within the invention is a method of inhibiting T cell or Bcell proliferation in a human, the method comprising indentifying ahuman in need of such inhibition, and administering to the human acompound capable of specifically inhibiting monocarboxylate transportwithin a T cell or B cell.

[0048] In another method for treating a patient suffering from animmune-mediated disorder or cancer, the method comprises administeringto the patient an effective amount of a compound that specificallyreduces expression of an MCT, the compound being selected from the groupconsisting of an anti-sense molecule, a ribozyme molecule, an RNAimolecule, and a triple helix forming molecule.

[0049] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer, the methodcomprising administering an effective amount of a pharmaceuticalcomposition comprising an MCT inhibitor identifiable or identified by

[0050] (i) contacting a monocarboxylate transporter protein with a testcompound under conditions suitable for binding; and

[0051] (ii) detecting specific binding of the compound to thetransporter protein; to a subject in need thereof

[0052] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer, comprising

[0053] (i) contacting a monocarboxylate transporter protein with a testcompound under conditions suitable for binding;

[0054] (ii) detecting specific binding of the compound to thetransporter protein;

[0055] (iii) preparing a pharmaceutical composition comprising thecompound; and,

[0056] (iv) administering an effective amount of the pharmaceuticalcomposition to a subject in need thereof.

[0057] According to another aspect of the invention there is providedthe use of an MCT inhibitor compound that decreases the activity of amonocarboxylate transporter, other than MCT1 and MCT2, in the treatmentof an immune-mediated disorder or cancer. The compound preferably doesnot significantly inhibit MCT1 or MCT2.

[0058] According to another aspect of the invention there is providedthe use of an MCT inhibitor compound that decreases the activity of amonocarboxylate transporter selected from the group consisting of MCT3and MCT4, in the treatment of an immune-mediated disorder or cancer. Thecompound preferably does not significantly inhibit MCT1 or MCT2.

[0059] According to another aspect of the invention there is providedthe use of a compound that inhibits or otherwise decreases the activityof a monocarboxylate transporter, other than a compound of Formulae I toIX or as disclosed in Michne et al., supra, or Baine et al., supra., inthe manufacture of a medicament for the treatment of an immune-mediateddisorder or cancer.

[0060] According to another aspect of the invention there is providedthe use of an MCT inhibitor compound that decreases the activity of amonocarboxylate transporter other than MCT1 and MCT2, in the manufactureof a medicament for the treatment of an immune-mediated disorder orcancer.

[0061] According to another aspect of the invention there is providedthe use of an MCT inhibitor compound that decreases the activity of amonocarboxylate transporter selected from the group consisting of MCT3and MCT4, in the manufacture of a medicament for the treatment of animmune-mediated disorder or cancer.

[0062] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer, comprising (i)in vitro testing a compound for the ability to inhibit lactate transportin a cell, and (ii) administering, to a human patient in need oftreatment, an effective amount of a compound which has been identifiedfrom step (i) as a compound capable of blocking lactate transport.

[0063] According to another aspect of the invention there is provided amethod of treating an immune-mediated disorder or cancer, comprising (i)testing a compound for its ability to inhibit monocarboxylate transportin a cell, and (ii) administering to a human patient suffering from orlikely to suffer from an immune-mediated disorder or cancer, of aneffective amount of a compound which has been identified from step (i)as a compound capable of inhibiting monocarboxylate transport.

[0064] According to another aspect of the invention there is provided amethod of treating a patient suffering from an immune-mediated disorderor cancer, comprising administering to a human patient suffering from orlikely to suffer from such a disease or condition of an effective amountof a compound which has been shown (or is known) to be capable ofblocking or inhibiting cellular monocarboxylate transport.

DETAILED DESCRIPTION

[0065] Potential therapeutic agents that may be tested in the screeningmethods described herein include simple organic molecules, commonlyknown as “small molecules”, for example those having a molecular weightof less than 2000 Daltons. Other potential therapeutics include peptidesand antibodies. The methods of the invention may be used to screen, forexample, chemical compound libraries or peptide libraries, includingsynthetic peptide libraries and peptide phage libraries, particularlyantibody display (such as scFV or Fab) phage libraries. Other suitablecompound molecules include antibodies, nucleotide sequences, and anyother molecules, including nucleic acid or polypeptide mimetics, thatbind to an MCT. Preferably the compound is a small molecule chemicalcompound. The terms compound and agent are used interchangeably herein.

[0066] The screening methods of the invention will prove useful indetermining whether or not test compounds (chemical or biological) maybe suitable for use, inter alia, in the treatment, includingprophylactic treatment, of cancers; autoimmune, inflammatory,proliferative and hyperproliferative diseases; and other immune-mediateddiseases including rejection of transplanted organs or tissues. Examplesof immune-mediated disorders and cancers include:

[0067] (1) (the respiratory tract) reversible obstructive airwaysdiseases including asthma, such as bronchial, allergic, intrinsic,extrinsic and dust asthma, particularly chronic or inveterate asthma(e.g., late asthma and airways hyper-responsiveness); bronchitis; acute,allergic, atopic and chronic rhinitis, including rhinitis caseosa,hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and rhinitismedicamentosa; membranous rhinitis including croupous, fibrinous,pseudomembranous and scrofoulous rhinitis; seasonal rhinitis includingrhinitis nervosa (hay fever) and vasomotor rhinitis; sarcoidosis,farmer's lung and related diseases, fibroid lung and idiopathicinterstitial pneumonia;

[0068] (2) (bone and joints) rheumatoid arthritis, seronegativespondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis and Reiter's disease), Behcet's syndrome, Sjogren's syndromeand systemic sclerosis;

[0069] (3) (skin) psoriasis, atopic dermatitis, contact dermatitis andother eczmatous dermitides, seborrhoetic dermitis, Lichen planus,Pemphigus, bullous Pemphigus, Epidermolysis bullosa, urticaria,angiodermas, vasculitides, erythemas, cutaneous eosinophilias, uveitis,Alopecia areata and vernal conjunctivitis;

[0070] (4) (gastrointestinal tract) Coeliac disease, proctitis,eosinophilic gastro-enteritis, mastocytosis, Crohn's disease, ulcerativecolitis, and food-related allergies that have effects remote from thegut, e.g., migraine, rhinitis and eczema;

[0071] (5) (other tissues and systemic disease) multiple sclerosis,atherosclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis,myasthenia gravis, type I (but not type II) diabetes, nephroticsyndrome, eosinophilia fascitis, hyper IgE syndrome, lepromatousleprosy, Sezary syndrome and idiopathic thrombocytopenia purpura;

[0072] (6) (allograft rejection) acute and chronic allograft rejectionfollowing, for example, transplantation of kidney, heart, liver, lung,bone marrow, skin, pancreatic islet cells, cornea and stem cells; andchronic graft versus host disease.

[0073] (7) (xenograft rejection) Hyperacute, acute and chronic xenograftrejection following, for example, transplantation of kidney, heart,liver, lung, bone marrow, skin, pancreatic islet cells, cornea and stemcells; and chronic graft versus host disease.

[0074] (8) (cancer) carcinoma, including that of the bladder, breast,colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroidand skin; hematopoietic tumors of lymphoid lineage, including acutelymphocytic leukemia, B cell lymphoma and Burketts lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; tumors of mesenchymalorigin, including fibrosarcoma and rhabdomyosarcoma; and other tumors,including melanoma, seminoma, tetratocarcinoma, neuroblastoma andglioma.

[0075] The compounds are thus indicated for use in the treatment orprevention of rejection of transplanted organs, tissues, or cells suchas kidney, heart, lung, bone marrow, skin, pancreatic islet cells,cornea and stem cells; and of autoimmune, inflammatory, proliferative,and hyperproliferative diseases, including cancer (preferably other thanCNS cancers, and more particularly other than glial cell cancers), andof cutaneous manifestations of immune-mediated disorders: for example,rheumatoid arthritis, systemic lupus erythematosus, Hashimoto'sthyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes,uveitis, nephrotic syndrome, psoriasis, atopic dermatitis, contactdermatitis and further eczematous dermatitides, seborrhoeic dermatitis,Lichen planus, Pemphigus, Epidermolysis bullosa, urticaria, angioedemas,vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata,eosinophilic fasciitis and atherosclerosis.

[0076] The inventors have found that compounds that are capable ofbinding to MCT1 and/or MCT2, and that inhibit lactate transport ofactivated T cells, inhibit the proliferation of activated T-cells andtumour cell lines such as the erythroleukaemia cell line K562. The firstaspect of the invention is therefore a screening method to identifycompounds that may be useful, inter alia, in treating conditions ordiseases involving T-cell activation, such as transplant rejection andrheumatoid arthritis, or cellular proliferation, such as cancer.

[0077] In one embodiment the compound is tested for its ability toinhibit MCT activity. This, for example, may be via inhibition of theability to transport monocarboxylates or via blockage of MCT expression.It is well known that MCT proteins from different species have a highdegree of sequence similarity. For example, rat MCT1 is reported topossess 86% identity with human MCT1 (Jackson et al., Biochem BiophysActa 1238:193-196, 1995). Thus, whilst in a preferred embodiment the MCTis of human origin, particularly from the group consisting of humanMCT1, 2, 3 and 4, it is envisaged that MCTs from other species such asrat or mouse would also work in the invention. Indeed, the inventorshave found that the screening method works equally well using rat MCT1protein. Suitable monocarboxylate transporters for use in the screeningassay/method of the invention include MCT1, MCT2 and MCT4.

[0078] The sequence of MCT1 is disclosed in the EMBL/GenBank/DDBJdatabases (Blum H., Bauersachs S., Mewes H. W., Weil B., Wiemann S,Submitted (15 Mar. 2000) to the EMBL/GenBank/DDBJ databases) with theEMBL Accession No. AL162079. The sequence of the cDNA clone encodinghuman MCT1 used herein is disclosed in SEQ ID NO: 1, and is identical tothe sequence disclosed by Blum et al. (supra).

[0079] With regard to MCT2, there appears to be no single definitivepublished sequence. Two MCT2 sequences deposited in EMBL (AccessionNumbers AF049608 and AF058056) differ in three locations that lead toamino acid changes. The sequence of the cDNA clone encoding human MCT2used herein, and disclosed in SEQ ID NO: 2, is a combination of the two.The specific amino acid differences are as follows: (i) AF049608 encodesthe amino acid Asparagine at position 154, whilst AF058056 and SEQ IDNO:2 both encode a Serine at this position; (ii) AF049608 encodes theamino acid Proline at position 268, whilst AF058056 and SEQ ID NO:2 bothencode a Leucine at this position; and (iii) AF058056 encodes the aminoacid Serine at position 445, whilst AF049608 and SEQ ID NO:2 both encodea Threonine at this position. There are no differences between SEQ IDNO:2 and the MCT2 genomic exon and predicted transcript sequences (UCSCSOFTBERRY Database Accession No. C12001042), which confirms that theMCT2 cDNA clone depicted in SEQ ID NO:2 is native.

[0080] With regard to MCT3, compared to the predicted MCT3 amino acidsequence disclosed in Yoon et al. (Genomics. 60(3):366-370, 1990), thesequence used herein (SEQ ID NO:3) has an amino acid substitution ofTryptophan to Arginine at position 235. The published MCT3 genomicsequence (Accession No. AL031587) is consistent with the sequence of SEQID NO:3, encoding an Arginine at position 235.

[0081] The screening assay is not restricted to use of the full-lengthMCT proteins as represented in Table 1, but extends to functionalvariants, including mutants, deletions and chimaeric variants thatmaintain activity in the test assay. TABLE 1 Human MCT ReferenceAccession No. MCT1 Blum H., Bauersachs S., Mewes H. W., Weil B., EMBLAL162079 Wiemann S, Submitted (15-MAR-2000) to the EMBL/GenBank/DDBJdatabases MCT2 Journal of Biological Chemistry 273 (44), 28959- EMBLAF049608 28965 (1998) Lin, R. Y., Vera, J. C., Chaganti, R. S. K. andGolde, D. W. Human monocarboxylate transporter 2 (MCT2) is a highaffinity pyruvate transporter. Dao L., Landschulz W. H., Landschulz K.T.; EMBL AF058056 “Cloning of Human Monocarboxylate Transporter 2(hMCT2)”; Unpublished. Submitted (07-APR- 1998) to the EMBL/GenBank/DDBJdatabases. Genomic and predicted transcript sequence for UCSC SOFTBERRYMCT2; UCSC SOFTBERRY Database Accession C12001042 No. C12001042 MCT3Genomics 60 (3), 366-370 (1999) EMBL AF132610 Yoon, H., Donoso, L. A.and Philp, N. J. Cloning of the human monocarboxylate transporter MCT3gene: localization to chromosome 22q12.3- q13.2. Phillimore B.;Submitted (08-DEC-1999) to the EMBL AL031587 EMBL/GenBank/DDBJ databasesMCT4 Biochem. J 329 (Pt 2), 321-328 (1998) EMBL U81800 Price, N. T.,Jackson, V. N. and Halestrap, A. P. Cloning and sequencing of four newmammalian monocarboxylate transporter (MCT) homologues confirms theexistence of a transporter family with an ancient past.

[0082] Each of these publications is incorporated herein by reference.

[0083] The invention is not restricted to the use of full-length nativehuman MCTs. The use of suitable functional variants also forms part ofthis invention. In particular, included within the scope of the presentinvention is use of alleles of the disclosed MCT proteins. As usedherein, an “allele” or “allelic sequence” is a naturally occurringalternative form of the molecule described herein. Alleles result fromnucleic acid mutations and mRNA splice-variants, which producepolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one or many allelic forms. Common mutationalchanges that give rise to alleles are generally ascribed to naturaldeletions, additions or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

[0084] Also useful in the invention are artificially producedpolypeptides that share sequence homology with, and consequently havesubstantially the same ligand binding ability or monocarboxylatetransport activity as, the proteins coded for by the nucleotide sequencedepicted in SEQ ID Nos 1 to 4. Within this category are truncated ormutated versions of the disclosed MCT proteins and chimeric polypeptidesincluding some MCT sequence and some heterologous sequence. Suchpolypeptides will preferably be substantially homologous to thedisclosed MCT proteins. By the term “substantially homologous,” we meana sequence that possesses at least 70%, and in increasing order ofpreference at least 75%, 80%, 85%, 90%, 95%, 97% and 99% sequenceidentity thereto. By the term “substantially the same biologicalactivity,” we mean having the ability to effect monocarboxylatetransport. In a preferred embodiment the variant proteins will have atleast 10%, and in increasing order of preference at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% and 95% of the monocarboxylate transportationactivity of the respective wild-type protein when recombinantlyexpressed in a suitable heterologous expression system.

[0085] The sequence identity between two sequences can be determined bypair-wise computer alignment analysis, using the BestFit program. Inpractice, when searching for similar/identical sequences to the querysearch, from within a sequence database, it is generally necessary toperform an initial identification of similar sequences using suitablesoftware such as Blast, Blast2, NCBI Blast2, WashU Blast2, FastA,Fasta3, PILEUP and CLUSTALW, and a scoring matrix such as Blosum 62.Such software packages endeavour to closely approximate the“gold-standard” alignment algorithm of Smith-Waterman. Thus, thepreferred software/search engine programme for use in assessingsimilarity, i.e., how two primary polypeptide sequences line up, isSmith-Waterman. Identity refers to direct matches, similarity allows forconservative substitutions.

[0086] As used herein, the term “isolated” refers to molecules, eithernucleic acid or amino acid sequences, that (1) are removed from theirnatural environment and purified or separated from at least one othercomponent with which they are naturally associated, or (2) areartificially synthesised, or (3) are recombinantly produced in a milieuin which they do not naturally occur (e.g., a human MCT proteinexpressed in a non-human cell, or in a human cell different from a typeof cell that normally expresses it); or (4) possess a sequence thatdiffers from any known naturally occurring sequence.

[0087] Although the natural polypeptide of SEQ ID NO. 1 and a variantpolypeptide may only possess for example 80% identity, they are actuallylikely to possess a higher degree of similarity, depending on the numberof dissimilar codons that are conservative changes. Similarity betweentwo sequences includes direct matches as well as conserved amino acidsubstitutes that possess similar structural or chemical properties,e.g., similar charge. Examples of conservative changes (conservativeamino acid substitutes) are shown in Table 2.

[0088] Suitable conservative substitutions of amino acids are known tothose of skill in this art and generally may be made withoutsignificantly altering the biological activity of the resultingpolypeptide, regardless of the chosen method of synthesis. The phrase“conservative substitution” includes the use of a chemically derivatizedresidue in place of a non-derivatized residue, provided that theresulting polypeptide displays the desired binding activity. D-isomersas well as other known derivatives may also be substituted for thenaturally occurring amino acids. See, e.g., U.S. Pat. No. 5,652,369,Amino Acid Derivatives, issued Jul. 29, 1997. Substitutions arepreferably, although not exclusively, made in accordance with those setforth in TABLE 2 as follows: TABLE 2 Original residue Exampleconservative substitution Ala (A) Gly; Ser; Val; Leu; Ile; Pro Arg (R)Lys; His; Gln; Asn Asn (N) Gln; His; Lys; Arg Asp (D) Glu Cys (C) SerGln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln; Arg; Lys Ile(I) Leu; Val; Met; Ala; Phe Leu (L) Ile; Val; Met; Ala; Phe Lys (K) Arg;Gln; His; Asn Met (M) Leu; Tyr; Ile; Phe Phe (F) Met; Leu; Tyr; Val;Ile; Ala Pro (P) Ala; Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr(Y) Trp; Phe; Thr; Ser Val (V) Ile; Leu; Met; Phe; Ala

[0089] The MCT coding nucleotide sequences for use in the presentinvention may also be engineered in order to alter a coding sequence fora variety of reasons, including but not limited to alterations thatmodify the cloning, processing and/or expression of the gene product.For example, mutations may be introduced using techniques that are wellknown in the art, e.g., site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns, to change codonpreference, etc.

[0090] Because the MCT proteins are predicted to have a transmembranedomain structure, they will likely require a membrane scaffold to retaintheir structural and/or functional integrity. Whilst the preferred assaymethods involve use of whole cells, or cell membrane preparationsthereof, that contain one or more MCTs, it will be appreciated that theMCT proteins or polypeptides can be presented in alternate formats toretain their structural integrity. For example, the proteins may bereconstituted within lipid vesicles (see, for example, Lynch andMcGiven, Biochem. J 244:503-508, 1987). Such alternate means ofpresenting the monocarboxylate transporter protein are also part of theinvention. Whole cells expressing MCT or membrane preparations thereofare particularly useful. Suitable whole cells may either be naturalcells or cell lines that comprise endogenous MCTs, such as Jurkat, K562,HeLa, and Chinese Hamster Ovary (CHO) cells, or transformed/transfectedcells such as INS1 and SF9 cells, wherein the MCT protein has beenintroduced via recombinant techniques well known to the person skilledin the art.

[0091] In one embodiment, cells or cellular membrane preparationscontaining an MCT protein derived from cells, preferably eukaryotic,particularly mammalian, transformed, transfected or transduced with arecombinant expression construct comprising the nucleotide sequencecoding for an MCT protein and sequences sufficient to direct thesynthesis of the MCT protein in cultures of said transformed,transfected or transduced cells, are used to determine the bindingproperties of test compounds in vitro.

[0092] In one particular embodiment, the MCT protein is expressed ineukaryotic cells, especially mammalian, insect or yeast cells.Eukaryotic cells provide post-translational modifications torecombinantly expressed proteins, including folding and/orphosphorylation and/or glycosylation.

[0093] Nucleic acids coding for an MCT for use in the invention can beeither isolated (e.g., from a cDNA library) or synthesised, and avariety of expression vector/host systems may be used to express MCTcoding sequences. These include but are not limited to microorganismssuch as bacteria transformed with plasmids, cosmids or bacteriophage;yeast transformed with expression vectors; insect cells transformed witheither the baculovirus expression system or insect expression plasmids;plant cells transfected with plant virus expression systems, such ascauliflower mosaic virus; or mammalian cell systems (for example thosetransfected or transduced with plasmid or viral derived expressionvectors, e.g., retroviral, pox, or adenoviral vectors). Selection of themost appropriate system is a matter of choice.

[0094] Expression vectors usually include an origin of replication, apromoter, a translation initiation site, optionally a signal peptide, apolyadenylation site, and a transcription termination site. Thesevectors also usually contain one or more antibiotic resistance markergene(s) for selection. As noted above, suitable expression vectors maybe plasmids, cosmids or viruses such as phage or retroviruses. Thecoding sequence of the polypeptide is placed under the control of anappropriate promoter, control elements and transcription terminator sothat the nucleic acid sequence encoding the polypeptide is transcribedinto RNA in the host cell transformed or transfected by the expressionvector construct. The coding sequence may or may not contain a signalpeptide or leader sequence for secretion of the polypeptide out of thehost cell; in general, signal peptides do not appear necessary for MCTproteins that end up imbedded in the cellular membrane. Preferredvectors will usually comprise at least one multiple cloning site tofacilitate cloning of the gene.

[0095] Methods for the expression of MCT proteins in host cells fromcloned genes is well known to those skilled in the art of molecularbiology and general techniques are described in such publications asMolecular Cloning—A Laboratory Manual, Second Edition, Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory, 1989) and Current Protocolsin Molecular Biology, Volumes1-3, Edited by F M Asubel, R Brent, R EKingston (pub John Wiley 1998). Examples of host cells that may betransformed or transfected with nucleic acid encoding an MCT protein soas to express said MCT protein include prokaryotic cells, e.g.,bacterial cells such as Escherichia coli and Bacillus subtilis; lowereukaryotic cells, e.g., yeasts such as Saccharomyces cerevisiae,Schizosaccharmoyces pombe, Pichia pastoris, Candida albicans,Aspergillus nidulans or Neurospora crassa; higher eukaryotic cells,e.g., mammalian cells such as CHO, NIH-3T3, HEK-293, Jurkat, and INS-1;insect cells such as Spodoptera frugiperda 9 and 21 cell lines; andamphibian cells such as Xenopus laevis oocytes. Performance of theinvention is neither dependent on nor limited to any particular strainor type of host cell or vector; those suitable for use in the inventionwill be apparent to, and a matter of choice for, the person skilled inthe art.

[0096] Host cells transformed or transfected with a vector containing anMCT nucleotide sequence may be cultured under conditions suitable forexpression and recovery of membrane fractions containing the encodedproteins from the cell culture. Such expressed proteins will preferably,but not necessarily, be presented on the cell surface. Native cellslines used for detecting binding to MCTs or functional activity in MCTsinclude, e.g., K562 (human erythroleukaemia cell line) and MB231 (breastcarcinoma cell line). Primary cells that naturally express MCTs may alsobe used for detecting binding to MCTs or functional activity in MCTs.Such primary cell types are known in the art; others can be identifiedby, e.g., immunoaffinity, Western blots, or Northern blots.

[0097] Membrane preparations for use in the invention can be made usingstandard techniques well known to the person skilled in the art,including the method disclosed in Example 10.

[0098] It will be appreciated that there are many screening methods thatmay be employed to determine the ability of a test compound to decreasemonocarboxylate transport. Indeed, monocarboxylate transport activitycan be measured directly or indirectly in a number of ways that will beapparent to the person skilled in the art. This invention incorporateseach of these different ways. For example, direct binding to an MCT,such as MCT1 protein, can be determined by standard ligand bindingassays. Such assays can be performed using whole cells or cell membranepreparations containing MCT proteins. Suitable alternative assays mightmeasure monocarboxylate accumulation within the cell, monocarboxylateefflux from the cell, H+ efflux or accumulation, alterations in theglycolytic rate due to monocarboxylate feedback regulation, decreasedDNA synthesis and/or cell division, and the like. Examples of suitablescreening methods that may be used to identify an inhibitor ofmonocarboxylate transport include rapid filtration of equilibriumbinding mixtures, radioimmunoassays (RIA) and fluorescence resonanceenergy transfer assays (FRET). A particularly useful method foridentifying a compound capable of inhibiting monocarboxylate transportis a scintillation proximity assay (SPA).

[0099] SPA involves the use of fluoromicrospheres coated with acceptormolecules, such as receptors, to which a ligand will bind selectively ina reversible manner (N Bosworth & P Towers, Nature 341:167-168, 1989).The technique requires the use of a ligand labelled with an isotope thatemits low energy radiation that is dissipated easily into an aqueousmedium. At any point during an assay, bound labelled ligands will be inclose proximity to the fluoromicrospheres, allowing the emitted energyto activate the fluor and produce light. In contrast, the vast majorityof unbound labelled ligands will be too far from the fluoromicrospheresto enable the transfer of energy. Bound ligands produce light but freeligands do not, allowing the extent of ligand binding to be measuredwithout the need to separate bound and free ligand.

[0100] The following disclosure of suitable screening methods is merelyintended to be an overview, and is not intended to reflect the fullstate of the art. Measurement of lactate efflux/accumulation by:

[0101] 1) Enzymatic measurement of lactate levels using lactate as asubstrate for lactate oxidase or lactate dehydrogenase usingcommercially available kits such as the Sigma LO kit (735-10) or SigmaLD kit (826); or a glucose/lactate analyser (YSI 2700 analyser).

[0102] 2) Transport of [¹⁴C]lactate (or another radiolabelled substrateof the monocarboxylate transporter, e.g., pyruvate, β-hydroxybutyrate,or glycolate) in an assay such as that described by Poole and Halestrapin Am. J. Physiol. 264:C761-C782 (1993).

[0103] 3) Lactate-induced decrease in intracellular pH using pHsensitive dyes, e.g., 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein(BCECF), in an assay such as that described by Carpenter and Halestrapin Biochem. J 304:751-760 (1999).

[0104] 4) Lactate-induced decrease in intracellular pH usingpH-sensitive electrodes in an assay such as that described by Bröer etal. in Biochem. J 333:167-174 (1998).

[0105] 5) Measurement of proton efflux/accumulation using amicrophysiometer in an assay such as that described, for example, byMcConnell et al., Science 257(5078):1906-1912 (1992). Each of thesepublications is incorporated herein by reference.

[0106] In one embodiment, the screening assay method is a competitivebinding assay. Thus, according to a further aspect of the inventionthere is provided a competitive binding assay for compounds that mayhave potential in treating an immune-mediated disorder or cancer, whichcomprises contacting host cells expressing MCT protein, or a membranepreparation thereof, with both a first test compound and a labelledsecond compound known to specifically bind to said MCT protein, underconditions suitable for binding of both compounds, and detectingspecific binding of the first compound to the MCT protein by measuring adecrease in the binding of the second compound to the MCT protein in thepresence of the first compound, indicating that the first compound bindsto the MCT protein and may thus have potential in treating animmune-mediated disorder or cancer.

[0107] As a comparative control, the assay can be conducted with onlythe second compound.

[0108] The first compound is preferably a small molecule compound andthe second compound is preferably either a small molecule compound or anantibody. (The term “small molecule compound” is given its standardmeaning in the pharmaceutical industry; it generally is meant to excludelarge biological molecules such as polypeptides and nucleic acids.Typically “small molecule compounds” are at least partially chemicallysynthesized.) In a further embodiment, the MCT is a human MCT. In afurther embodiment the MCT is selected from the group consisting ofMCT1, 2, 3, and 4. Preferably, the MCT is human MCT1.

[0109] There are many conventional detectable labels, such asradioisotopes, fluorescent labels, chemiluminescent compounds, labelledbinding proteins, magnetic labels, spectroscopic markers and linkedenzymes that might be used to label up the second compound. Fluorescentlabels are often preferred because they are less hazardous thanradiolabels, they provide a strong signal with low background, andvarious different fluorophors capable of absorbing light at differentwavelengths and/or giving off different colour signals exist to enablecomparative analysis in the same analysis. For example, fluoresceingives off a green colour, rhodamine gives off a red colour, and bothtogether give off a yellow colour. For use in the present invention,preferred labels are radioisotopes, particularly ¹⁴C, ³H and ¹²⁵I, ornon-radioactive labels such as digoxigenin or biotin. The choice oflabel and the means of detecting such label (such as via autoradiographyor fluorescence microscopy) can be made by the person skilled in theart.

[0110] In one embodiment of the invention a radioligand binding assay isperformed, which comprises contacting the test compound with a cellularmembrane preparation containing an MCT, preferably MCT1, and aradio-labelled ligand that is known to bind to said MCT, and measuringdisplacement of said ligand by the test compound.

[0111] In one embodiment, the test compounds will be specific for aparticular MCT subtype. Compounds are deemed specific if they bind toone particular MCT subtype and exhibit at least 10-fold lower potency(Ki), preferably at least 25-fold lower potency, and more preferably atleast 100-fold lower potency to all other subtypes.

[0112] Potential drug candidates are identified by choosing chemicalcompounds that bind with high affinity (IC50 of less than 10 μM) to theexpressed MCT, by using, for example, ligand binding methods well knownto those skilled in the art, examples of which are shown in the bindingassays described herein. Drug candidates may have broad specificityacting on more than one MCT subtype; alternatively, the drug candidateswill be specific for a particular MCT subtype. Compounds are deemedspecific if they inhibit monocarboxylate transport by one particular MCTsubtype at least ten fold, preferably at least 25 fold, and morepreferably at least 100 fold more strongly than any other MCT subtype.Alternatively, compounds may be deemed specific if they bind at leastten fold, preferably at least 25 fold and more preferably at least 100fold more strongly to one particular MCT subtype than to any other MCTsubtype.

[0113] Ligands A and C (described in Example 1) are examples of suitableradioligands that can be used in the invention. Such radioligands can bemade by standard techniques. These radioligands are a further aspect ofthe invention.

[0114] Thus, according to a further aspect of the invention there isprovided a radiolabeled compound capable of binding to an MCT. In termsof a radioligand for binding to human MCT1, any of the compoundsdisclosed in any of WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695,WO 00/12514, WO 01/83489; PCT/GB02/03399, PCT/GB02/03250; andGB-A-2363377 could be used. In one embodiment, the radiolabelledcompound is selected from the group consisting of ligands A, B and C (asdescribed in Example 1 herein). In another aspect of the invention,there is provided the use of a radiolabelled compound capable of bindingto an MCT in a screening assay to identify compounds capable of bindingsaid MCT. In one embodiment, the MCT is MCT1, in another, theradiolabelled compound is ligand A, B or C, as described herein.

[0115] The MCT proteins or convenient fragments thereof may be used toraise antibodies. Such antibodies have a number of uses that will beevident to the molecular biologist or immunologist of ordinary skill.Such uses include, but are not limited to, use as a biotherapeutic, useas the competitive binding ligand in the screening methods of theinvention, and monitoring protein expression. Enzyme linkedimmunosorbant assays (ELISAs) are well known in the art and would beparticularly suitable for detecting the MCT polypeptide or fragmentsthereof. The term antibody includes both monoclonal antibodies, whichare a substantially homogeneous population, and polyclonal antibodies,which are heterogeneous populations. The term also includes, inter alia,humanised and chimeric antibodies, as well as the various types ofantibody constructs such as for example F(ab′)₂, Fab and single chainFv, including bacteriophage derived antibodies.

[0116] In one embodiment, such antibodies are labelled. Methods ofmaking and detecting labelled antibodies are well known (Campbell;Monoclonal Antibody Technology, in: Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13. Eds: Burdon R et al.Elsevier, Amsterdam (1984)).

[0117] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well known in the art.In general, antigen is administered to the host animal, typicallythrough parenteral injection. Depending on the host species, variousadjuvants may be used to enhance the immunological response against theinjected polypeptide. Suitable adjuvants include but are not limited toFreund's (complete and incomplete), aluminium hydroxide, BCG and SAC(Bacille Calmette-Guerin and Staphylococcus aureus Cowan). Followingbooster immunizations, small samples of serum are collected and testedfor reactivity to antigen. Examples of various assays useful for suchdetermination include those described in Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988; as well as procedures such as countercurrentimmuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immunosorbent assays (ELISA),dot blot assays, and sandwich assays (see, e.g., U.S. Pat. Nos.4,376,110 and 4,486,530).

[0118] Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011; 4,902,614; 4,543,439 and 4,411,993; and MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, PlenumPress, Kennett, McKearn, and Bechtol (eds.), (1980). By way of example,for the production of human monoclonal antibodies, hybridoma cells maybe prepared by fusing spleen cells from an immunised animal, e.g., amouse, with a tumour cell. Appropriately secreting hybridoma cells maythereafter be selected (Koehler & Milstein. Nature. 256:495-497, 1975;Cole et al. “Monoclonal antibodies and Cancer Therapy”, Alan R Liss Inc,New York N.Y. pp 77-96, 1985). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. Rodent antibodies may be humanised using recombinant DNAtechnology according to techniques known in the art.

[0119] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology (1990) 3:1-9, which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology (1989) 7: 394.

[0120] Alternatively, chimeric antibodies, single chain antibodies (seefor example, U.S. Pat. No. 4,946,778), or Fab fragments may also bedeveloped against the polypeptides utilized in the invention (Huse etal., Science. 256:1275-1281, 1989), using techniques known in the art.

[0121] Antibodies are defined to be specifically binding if they bindthe particular MCT with a K_(a) of greater than or equal to about 10⁷M⁻¹. Affinity of binding can be determined using conventionaltechniques, for example those described by Scatchard et al., Ann. N.Y.Acad. Sci., (1949) 51:660.

[0122] Once isolated and purified, the antibodies may be used to detectthe presence of antigen in a sample using established assay protocols,see for example “A Practical Guide to ELISA” by D. M. Kemeny, PergamonPress, Oxford, England. Methods of making and detecting labelledantibodies are well known (Campbell; Monoclonal Antibody Technology, in:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13.Eds: Burdon R et al. Elsevier, Amsterdam (1984)).

[0123] Method of Treatment

[0124] The inventors' radioligand studies indicate that the actualmechanism of action by which the compounds in International PublicationNos. WO 98/46606, WO 98/54190, WO 98/28301, WO 99/29695, WO 00/12514,and WO 01/83489; International Application numbers PCT/GB02/03399 andPCT/GB02/03250 and GB-A-2363377, each incorporated herein by reference,operate, is via binding to and inhibiting MCT1 and to a lesser extentMCT2, leading inter alia, to a buildup of lactate in the cell.

[0125] The compounds identified in these prior art patents fall withinthe scope of one or other of Formulae I to IX:

[0126] in which:

[0127] R¹ is C₁₋₆alkyl, C₃₋₆alkenyl or C₃₋₆cycloalkyl;

[0128] R² is C₁₋₄alkyl or C₃₋₆alkenyl;

[0129] R³ is 1- or 2-indanyl, 1- or 2-(1,2,3,4-tetrahydronaphthalenyl),9-fluorenyl, acenaphthyl or CHR⁴(CH₂)_(n)Ar where n is 0 or 1, R⁴ ishydrogen or C₁₋₆alkyl and Ar is quinolinyl, naphthalenyl,benzodioxolinyl optionally susbstituted by one or more halogen atoms, orphenyl optionally substituted by one or more substituent groups selectedfrom halogen, C₁₋₆alkyl, C₁₋₆alkoxy and phenylsulfonylmethyl;

[0130] W is H, CH₂OH, CO₂H, CO₂C₁₋₆alkyl, CH₂NR⁵R⁶, CONR⁵R⁶, where R⁵and R⁶ are independently hydrogen or C₁₋₆alkyl, or together with thenitrogen atom to which they are attached form a 3- to 8-memberedheterocyclic ring optionally further containing an oxygen atom or agroup NR⁷ where R⁷is hydrogen or C₁₋₆alkyl, or W is pyridyl or phenyl,each of which may be optionally substituted by one or more substituentgroups selected from halogen, hydroxyl, C₁₋₆alkyl and C₁₋₆alkoxy;

[0131] X is a bond or C₁₋₅alkylene;

[0132] Y is S(O)_(p), C≡C, CH═CH, CH₂CH₂ or CH₂CH═CH; and

[0133] p is 0, 1 or 2;

[0134] or a pharmaceutically acceptable salt thereof, provided that:

[0135] X is not a bond when W is H, CH₂OH, CO₂H, CO₂C₁₋₆alkyl, CH₂NR⁵R⁶or CONR⁵R⁶ and Y is sulfur.

[0136] wherein:

[0137] R is —C(O)Ar¹, —C(R⁴)(R⁵)Ar¹, or Ar²;

[0138] Ar¹ is naphthyl, quinolyl, isoquinolyl, indolyl, benzofuranyl orbenzothienyl, each of which can be optionally substituted by one or moresubstituents selected from C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen ortrifluoromethyl, or Ar¹ is phenyl optionally substituted by one or moresubstituents selected from C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen,trifluoromethyl, amino, nitro, cyano, trifluoromethoxy, phenoxy,—CH₂N(R⁶)₂, —NHSO₂CF₃, C₁₋₄alkylsulphonylamino, —NHC(O)R^(6a), CO₂R⁷ or—C(O)NR⁸R^(8a);

[0139] R⁴ represents H or C₁₋₄ alkyl;

[0140] R⁵ represents H or OH;

[0141] each R⁶ independently represents H or C₁₋₄ alkyl;

[0142] R^(6a) represents H, C₁₋₆ alkyl, aryl or arC₁₋₄alkyl, wherein thearyl group or aryl moiety in the aralkyl group is phenyl or pyridyl,each of which may be optionally substituted by one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylcarbonylamino, halogenor trifluoromethyl;

[0143] R⁷represents H or C₁₋₄ alkyl;

[0144] R⁸ and R^(8a) each independently represent H, C₁₋₄ alkyl, phenylor pyridyl;

[0145] Ar² is acenaphthenyl, indanyl, iminodihydrobenzofuranyl orfluorenyl, each of which can be optionally substituted by one or moresubstituents selected from OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen, ortrifluoromethyl;

[0146] R¹ and R² are independently H, C₁₋₆ alkyl, C₃₋₆ alkenyl, CH₂C₃₋₅cycloalkyl or C₃₋₆ cycloalkyl;

[0147] R³represents H, X—R⁹ or X—Ar³;

[0148] X represents S(O)_(n), C(O)NR¹⁰, C(O)O, NH(CO)NR¹⁰, NH(CO)O orSO₂NR¹⁰;

[0149] n is 0, 1 or 2;

[0150] R⁹ represents a methyl group optionally substituted by one ormore substituents selected from CN, CO₂H, C₁₋₅ alkoxycarbonyl,5-tetrazolyl, SO₂NH₂ or C(O)NR¹¹R¹², or R⁹ represents C₂₋₆ alkyl or C₃₋₆alkenyl, each of which may be optionally substituted by one or moresubstituents selected from OH, CN, CO₂H, C₁₋₅ alkoxy, C₁₋₅alkoxycarbonyl, 5-tetrazolyl, azide, phthalimido, SO₂NH₂, C(O)NR¹¹R¹²,NR¹³R¹⁴, NHC(O)R¹⁵ or NHSO₂R¹⁶ where R¹¹, R¹², R¹³ and R¹⁴ eachindependently represent H or C₁₋₄ alkyl,

[0151] R¹⁵ represents C₁₋₄ alkyl, C₁₋₄ alkoxy, di(C₁₋₄alkyl)amino, oralkoxyalkylene containing up to 6 carbon atoms, and R¹⁶ represents C₁₋₄alkyl or trifluoromethyl; or, additionally, in the case where Xrepresents C(O)NR¹⁰, NH(CO)NR¹⁰ or SO₂NR¹⁰, R⁹ and R¹⁰ together with thenitrogen atom to which they are attached may form a 4- to 7-memberedheterocyclic ring which may be optionally substituted by one or more OHgroups;

[0152] R¹⁰ represents H, C₁₋₆ alkyl or is linked to R⁹ as defined above;and

[0153] Ar³ is phenyl, pyridyl or pyridine N-oxide, each of which may beoptionally substituted by one or more substituents selected from OH,NO₂, NH₂, NHSO₂CF₃, C₁₋₄ alkoxy, bis-C₁₋₄alkanesulphonylamino,C₁₋₄alkylcarbonylamino or C₁₋₄alkoxycarbonylamino; or apharmaceutically-acceptable salt or solvate thereof.

[0154] wherein B represents a group CH or a nitrogen (N), sulfur (S) oroxygen (O) atom; D represents a carbon (C) or nitrogen (N) atom; Erepresents a group CR³ or a nitrogen (N) atom; when D is a carbon atom,then B is a sulfur or oxygen atom and E is a group CR³, and when D is anitrogen atom, then either B is a group CH and E is a group CR³ or anitrogen atom, or B is a nitrogen atom and E is a group CR³; R¹represents a group NR′R″ where R′ represent a hydrogen atom or a C₁-C₆alkyl group, R″ represents a C₁-C₆ alkyl group, or R′ and R″ togetherwith the nitrogen atom to which they are attached form a 3- to7-membered saturated heterocyclic ring, or R¹ represents a C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₃-alkyloxyC₁-C₃-alkyl, C₃-C₆-cycloalkyloxyC₁-C₃-alkyl,C₃-C₆ alkenyl, phenyl, C₃-C₇ cycloalkyl, C₃-C₅ cycloalkylmethyl or C₃-C₇cycloalkenyl group, each of which may be optionally substituted by oneor more halogen atoms; R² represents a methyl group, or a C₂-C₆ alkylgroup optionally substituted by a C₁-C₆ alkoxy group other than in the1-position; R³ represents a hydrogen atom or a group X—R⁵ or X—Ar¹; Xrepresents a group —O—, S(O)_(n), SO₂N(R⁶) or C(═O)N(R⁶); n is 0, 1 or2; R⁵ represents an optionally substituted alkyl or alkenyl group, or,additionally, in the case where X represents SO₂N(R⁶) or C(═O)N(R⁶), R⁵and R⁶ together with the nitrogen atom to which they are attached mayform an optionally substituted 3- to 7-membered heterocyclic ring; Ar¹represents an optionally substituted phenyl or pyridyl group; R⁶represents a hydrogen atom, C₁-C₆ alkyl or is linked to R⁵ as definedabove; R⁴ represents a group CHR⁷Ar² or Ar³ or, additionally, in thecase where D represents a carbon atom, a group C(O)Ar² or CR⁷(OH)Ar²;Ar² represents an aryl or heteroaryl group which may be optionallysubstituted; Ar³ represents an acenaphthenyl, indanyl or fluorenylgroup, each of which may be optionally substituted; and R⁷ represents ahydrogen atom or a C₁-C₄ alkyl group; or a pharmaceutically-acceptablesalt or solvate thereof.

[0155] wherein:

[0156] W represents —CH₂— or a bond; Q represents Ar¹ or Ar²; in thecase where W represents —CH₂—, Q represents an aryl group Ar¹ whereinAr¹ represents naphthyl, phenyl, quinolyl, isoquinolyl, indolyl,benzofuranyl or benzothienyl; in the case where W represents a bond, Qrepresents an aryl group Ar² wherein Ar² represents acenaphthenyl,fluorenyl or indanyl; wherein the ring systems which Ar¹ and Ar²represent may all be optionally substituted by one or more substituentsselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen, or trifluoromethyl; R¹⁰represents X—(A)_(p)—Y; X represents S(O)_(n), C≡C, (CH₂)₂, CH═CH orCH₂CH═CH; n represents 0, 1 or 2; A represents C₁₋₆ alkylene; p is 0 or1; Y represents CN, OR¹¹, CO₂R¹², CONR¹³R¹⁴, NR¹⁵R¹⁶, NHSO₂R¹⁷, NHCOR¹⁸or an optionally substituted aryl or heteroaryl group, provided thatwhen X represents S(O)_(n), and Y is other than an optionallysubstituted aryl or heteroaryl group, then p is 1 and also provided thatwhen X represents S(O)_(n), p is 1 and Y represents OH, then n is not 0;R¹³ and R¹⁴ independently represent H, C₁₋₅ alkyl or phenyl, whichlatter group may be substituted by one or more substituents selectedfrom C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen, or CO₂R²¹; and R¹, R², R¹¹, R¹²,R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R²¹ independently represent H or C₁₋₅ alkyl; or apharmaceutically acceptable derivative thereof.

[0157] wherein:

[0158] R represents a group —C(O)Ar¹ or —C(R⁴)(R⁵)Ar¹;

[0159] Ar¹ represents a heterocyclic group comprising a total of from 5to 10 atoms which include from 1 to 3 heteroatoms independently selectedfrom nitrogen, oxygen and sulfur, which group Ar¹ may be optionallysubstituted by one or more substituents independently selected from oxo,hydroxyl, C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen, trifluoromethyl, amino,nitro, cyano, trifluoromethoxy, phenoxy, —CH₂N(R⁶)₂, —NHSO₂CF₃,C₁₋₄alkylsulfonylamino, —NHC(O)R^(6a), CO₂R⁷ or —C(O)NR⁸R^(8a), with theproviso that Ar¹ does not represent an optionally substitutedbenzofuranyl, benzothienyl, indolyl, quinolyl or isoquinolyl group;

[0160] R⁴represents a hydrogen atom or a C₁₋₄ alkyl group;

[0161] R⁵ represents a hydrogen atom or a hydroxyl group;

[0162] each R⁶ independently represents a hydrogen atom or a C₁₋₄ alkylgroup;

[0163] R^(6a) represents a hydrogen atom or a C₁₋₆ alkyl, aryl orarC₁₋₄alkyl group, wherein the aryl group or aryl moiety in the aralkylgroup is phenyl or pyridinyl, each of which may be optionallysubstituted by one or more substituents independently selected from C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylcarbonylamino, halogen or trifluoromethyl;

[0164] R⁷ represents a hydrogen atom or a C₁₋₄ alkyl group;

[0165] R⁸ and R^(8a) each independently represent a hydrogen atom or aC₁₋₄ alkyl, phenyl or pyridinyl group;

[0166] R¹ and R² each independently represent a hydrogen atom or a C₁₋₆alkyl, C₃₋₆ alkenyl, CH₂C₃₋₅ cycloalkyl or C₃₋₆ cycloalkyl group;

[0167] R³ represents a hydrogen atom or a group X—R⁹ or X—Ar²;

[0168] X represents an oxygen atom, S(O)_(n), C(O)NR¹⁰, C(O)O,NH(CO)NR¹⁰, NH(CO)O or SO₂NR¹⁰, with the proviso that when X representsan oxygen atom and R represents a group —C(R⁴)(R⁵)Ar¹, then R⁴ and R⁵both represent a hydrogen atom;

[0169] n is 0, 1 or 2;

[0170] R⁹ represents a methyl group optionally substituted by one ormore substituents independently selected from cyano, carboxyl, C₁₋₅alkoxycarbonyl, 5-tetrazolyl or C(O)NR¹¹R¹², or R⁹ represents a C₂₋₆alkyl or C₃₋₆ alkenyl group, each of which may be optionally substitutedby one or more substituents independently selected from hydroxyl, cyano,carboxyl, C₁₋₅ alkoxy, C₁₋₅ alkoxycarbonyl, 5-tetrazolyl, azido,phthalimido, SO₂NH₂, C(O)NR¹¹R¹², NR¹³R¹⁴, NHC(O)R¹⁵ or NHSO₂R¹⁶ whereR¹¹, R¹², R¹³ and R¹⁴ each independently represent a hydrogen atom or aC₁₋₄ alkyl group, R¹⁵ represents a C₁₋₄ alkyl, C₁₋₄ alkoxy, amino or(di)C₁₋₄alkylamino group or an alkoxyalkylene group containing up to 6carbon atoms, and R¹⁶ represents a C₁₋₄ alkyl or trifluoromethyl group;or, additionally, in the case where X represents C(O)NR¹⁰, NH(CO)NR¹⁰ orSO₂NR¹⁰, R⁹ and R¹⁰ together with the nitrogen atom to which they areattached may form a 4- to 7-membered saturated heterocyclic ring whichmay be optionally substituted by one or more hydroxyl groups;

[0171] R¹⁰ represents a hydrogen atom or a C₁₋₆ alkyl group or is linkedto R⁹ as defined above; and Ar² is phenyl, pyridinyl, thienyl, pyridoneor pyridine N-oxide, each of which may be optionally substituted by oneor more substituents independently selected from halogen, hydroxyl,nitro, amino, NHSO₂CF₃, C₁₋₄ alkyl, C₁₋₄ alkoxy,bis-C₁₋₄alkanesulfonylamino, C₁₋₄alkylcarbonylamino orC₁₋₄alkoxycarbonylamino;

[0172] or a pharmaceutically-acceptable salt or solvate thereof.

[0173] wherein:

[0174] R is —C(O)Ar¹, —C(R⁴)(R⁵)Ar¹ or Ar³;

[0175] Ar¹ represents a 5- to 10-membered aromatic ring system whereinup to 3 ring atoms may be heteroatoms independently selected fromnitrogen, oxygen and sulphur, the ring system being optionallysubstituted by one or more substituents independently selected from C₁₋₄alkyl, C₁₋₄ alkoxy, halogen, trifluoromethyl, oxo, nitro, cyano, NR⁶R⁷and —CH₂NR⁸R⁹; R¹ and R² each independently represent a hydrogen atom,C₁₋₆ alkyl, C₃₋₆ alkenyl, CH₂C₃₋₅ cycloalkyl or C₃₋₆ cycloalkyl;

[0176] R³ represents a group X—Ar²;

[0177] X represents a group S(O)_(n), C(O) or CH(OH);

[0178] n is 0, 1 or 2;

[0179] Ar² represents a 5- or 6-membered aromatic ring wherein up to 4ring atoms may be heteroatoms independently selected from nitrogen,oxygen and sulphur, the ring being optionally substituted by one or moresubstituents independently selected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylthio, halogen, trifluoromethyl, oxo, hydroxyl, amino, nitro, cyanoand benzyl;

[0180] R⁴ represents a hydrogen atom or C₁₋₄ alkyl;

[0181] R⁵represents a hydrogen atom or hydroxyl group;

[0182] R⁶ and R⁷ each independently represent a hydrogen atom or C₁₋₄alkyl, or together with the nitrogen atom to which they are attachedform a 5- to 7-membered saturated heterocyclic ring;

[0183] R⁸ and R⁹ each independently represent a hydrogen atom or C₁₋₄alkyl, or together with the nitrogen atom to which they are attachedform a 5- to 7-membered saturated heterocyclic ring; and

[0184] Ar³ represents acenaphthenyl, indanyl or fluorenyl, each of whichmay be optionally substituted by one or more substituents independentlyselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen or trifluoromethyl;

[0185] with the proviso that when X represents S(O)_(n), then Ar² doesnot represent pyridyl or thienyl; or a pharmaceutically acceptable saltor solvate thereof.

[0186] wherein:

[0187] R is —C(O)Ar¹, —C(R⁴)(R⁵)Ar¹ or Ar³;

[0188] Ar¹ represents a 5- to 10-membered aromatic ring system whereinup to 3 ring atoms may be heteroatoms independently selected fromnitrogen, oxygen and sulphur, the ring system being optionallysubstituted by one or more substituents independently selected from C₁₋₄alkyl, C₁₋₄ alkoxy, halogen, trifluoromethyl, oxo, nitro, cyano, NR⁶R⁷and —CH₂NR⁸R⁹;

[0189] R¹ and R² each independently represent a hydrogen atom, C₁₋₆alkyl, C₃₋₆ alkenyl, CH₂C₃₋₅ cycloalkyl or C₃₋₆ cycloalkyl;

[0190] R³ represents a group X—R¹⁰ or Ar²;

[0191] X represents a bond or a group NR¹¹;

[0192] Ar² represents a 5- or 6-membered aromatic ring wherein up to 4ring atoms may be heteroatoms independently selected from nitrogen,oxygen and sulphur, the ring being optionally substituted by one or moresubstituents independently selected from C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylthio, acetyl, halogen, trifluoromethyl, oxo, hydroxyl, amino,nitro, cyano and benzyl;

[0193] R⁴ represents a hydrogen atom or C₁₋₄ alkyl;

[0194] R⁵ represents a hydrogen atom or hydroxyl group;

[0195] R⁶ and R⁷ each independently represent a hydrogen atom or C₁₋₄alkyl, or together with the nitrogen atom to which they are attachedform a 5- to 7-membered saturated heterocyclic ring;

[0196] R⁸ and R⁹ each independently represent a hydrogen atom or C₁₋₄alkyl, or together with the nitrogen atom to which they are attachedform a 5- to 7-membered saturated heterocyclic ring;

[0197] R¹⁰ represents C₁₋₆ alkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl, each ofwhich may be optionally subsituted by one or more substituentsindependently selected from carboxyl, hydroxyl, —C(O)—R¹², C₃₋₆cycloalkyl, morpholinyl, —NR¹³R¹⁴, —SR¹⁵, —OR¹⁶, phenyl and halophenyl,or

[0198] R¹⁰ represents a C₃₋₆ cycloalkylcarbonyl, —C(O)CH₂CN,halophenylcarbonyl or trifluoromethylcarbonyl group;

[0199] R¹¹ represents a hydrogen atom or a C₁₋₆ alkyl group;

[0200] R¹² represents piperazinyl optionally substituted by a C₁₋₆ alkylgroup, or R¹² represents a group —NR¹⁷R⁸;

[0201] R¹³ and R¹⁴ each independently represent a hydrogen atom, or aC₁₋₄ alkyl, C₁₋₄ hydroxyalkyl or —C(O)—R¹⁹ group, or

[0202] R¹³ and R¹⁴, together with the nitrogen atom to which they areattached, form a 5- to 7-membered saturated heterocyclic ring which maybe optionally substituted by one or more substituents independentlyselected from C₁₋₄ alkyl, hydroxyl and oxo;

[0203] R¹⁵ and R¹⁶ each independently represent a 5- or 6-memberedaromatic ring wherein up to 4 ring atoms may be heteroatomsindependently selected from nitrogen, oxygen and sulphur, the ring beingoptionally substituted by one or more substituents independentlyselected from halogen atoms, cyano and C₁₋₄ alkyl;

[0204] R¹⁷ and R¹⁸ each independently represent a hydrogen atom, or aC₁₋₄ alkyl group optionally substituted by one or more substituentsindependently selected from halogen atoms and hydroxyl;

[0205] R¹⁹ represents a C₁₋₆ alkyl or C₃₋₆ cycloalkyl group, each ofwhich may be optionally substituted by a hydroxyl group; and

[0206] Ar³ represents acenaphthenyl, indanyl or fluorenyl, each of whichmay be optionally substituted by one or more substituents independentlyselected from C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen and trifluoromethyl;

[0207] or a pharmaceutically acceptable salt or solvate thereof.

[0208] wherein:

[0209] R¹ and R² each independently represent a C₁₋₆alkyl, C₃₋₆alkenyl,C₃₋₅cycloalkyl(C₁₋₃)methyl or C₃₋₆cycloalkyl; each of which may beoptionally substituted by 1 to 3 halogen atoms;

[0210] R³ represents a group —CON(R¹⁰)YR¹¹ or —SO₂N(R¹⁰)YR¹¹;

[0211] [wherein Y is O, S or NR₁₂ (wherein R¹² is hydrogen orC₁₋₆alkyl);

[0212] and R¹⁰ and R¹¹ are independently C₁₋₆alkyl optionallysubstituted by halo, hydroxy, amino, C₁₋₆alkylamino ordi-(C₁₋₆alkyl)amino];

[0213] Q is —CO— or —C(R⁴)(R⁵)— (wherein R⁴ represents a hydrogen atomor C₁₋₄alkyl and R⁵ represents a hydrogen atom or hydroxyl group);

[0214] Ar represents a 5- to 10-membered aromatic ring system wherein upto 4 ring atoms may be heteroatoms independently selected from nitrogen,oxygen and sulphur, the ring system being optionally substituted by oneor more substituents independently selected from C₁₋₄alkyl, C₁₋₄alkoxy,halogen, haloalkyl, dihaloalkyl, trihaloalkyl, hydroxyC₁₋₄alkyl,C₁₋₄alkoxyC₁₋₄alkyl, C₁₋₄alkylthio, C₁₋₄alkoxycarbonyl, C₂₋₄alkanoyl,oxo, nitro, cyano, —N(R⁶)R⁷ and —(CH₂)_(p)N(R⁸)R⁹, hydroxy,C₁₋₄alkylsulphonyl, C₁₋₄alkylsulphinyl, carbamoyl, C₁₋₄alkylcarbamoyl,di-(C₁₋₄alkyl)carbamoyl, carboxy;

[0215] p is 1 to 4;

[0216] R⁶ and R⁷ each independently represent a hydrogen atom,C₁₋₄alkanoyl or C₁₋₄alkyl, or together with the nitrogen atom to whichthey are attached form a 5- to 7-membered saturated heterocyclic ring;

[0217] R⁸ and R⁹ each independently represent a hydrogen atom,C₁₋₄alkanoyl or C₁₋₄ alkyl, or together with the nitrogen atom to whichthey are attached form a 5- to 7-membered saturated heterocyclic ring;

[0218] or a pharmaceutically acceptable salt or prodrug thereof.

[0219] wherein:

[0220] R¹ and R² each independently represent a C₁₋₆alkyl, C₃₋₆alkenyl,C₃₋₅cycloalkyl(C₁₋₃)methyl or C₃₋₆cycloalkyl; each of which may beoptionally substituted by 1 to 3 halogen atoms;

[0221] R³ is isoxazolidin-2-ylcarbonyl ortetrahydroisoxazin-2-ylcarbonyl wherein each ring is optionallysubstituted by one hydroxy group;

[0222] Q is —CO— or —C(R⁴)(R⁵)— (wherein R⁴ represents a hydrogen atomor C₁₋₄alkyl and R⁵ represents a hydrogen atom or hydroxyl group);

[0223] Ar represents a 5- to 10-membered aromatic ring system wherein upto 4 ring atoms may be heteroatoms independently selected from nitrogen,oxygen and sulphur, the ring system being optionally substituted by oneor more substituents independently selected from C₁₋₄alkyl (optionallysubstituted by 1,2 or 3 hydroxy groups), C₁₋₄alkoxy, halogen, haloalkyl,dihaloalkyl, trihaloalkyl, C₁₋₄alkoxyC₁₋₄alkyl, C₁₋₄alkylthio,C₁₋₄alkoxycarbonyl, C₂₋₄alkanoyl, oxo, thioxo, nitro, cyano, —N(R⁶)R⁷and —(CH₂)_(p)N(R⁸)R⁹, hydroxy, C₁₋₄alkylsulphonyl, C₁₋₄alkylsulphinyl,carbamoyl, C₁₋₄alkylcarbamoyl, di-(C₁₋₄alkyl)carbamoyl, carboxy;

[0224] p is 1 to 4;

[0225] R⁶ and R⁷ each independently represent a hydrogen atom,C₁₋₄alkanoyl or C₁₋₄alkyl, or together with the nitrogen atom to whichthey are attached form a 5- to 7-membered saturated heterocyclic ring;

[0226] R⁸ and R⁹ each independently represent a hydrogen atom,C₁₋₄alkanoyl or C₁₋₄ alkyl, or together with the nitrogen atom to whichthey are attached form a 5- to 7-membered saturated heterocyclic ring;

[0227] or a pharmaceutically acceptable salt or prodrug thereof.

[0228] At the filing dates of these applications the mechanism of actionof these compounds was not known and therefore is not taught in theseapplications/publications. Moreover, the newly identified mechanism ofaction is distinct from, indeed a radical change, from that acted on bycurrent immunosuppressive pharmaceutical compounds, such as cyclosporinA.

[0229] The medical treatment methods of the invention are particularlysuitable for treating rheumatoid arthritis and for use before, duringand after transplantation surgery, to prevent host rejection of thetransplanted tissue. Cancers treated using the methods of the inventionare preferably not CNS cancers such as glial cell cancers or cancersthat have metastasized to brain.

[0230] Compounds capable of effecting monocarboxylate build up in a cellor preventing monocarboxylate efflux from the cell are particularlysuitable for use in the disease treatment methods of the invention.

[0231] The compositions of the invention may be in a form suitable fororal use (for example as tablets, lozenges, hard or soft capsules,aqueous or oily suspensions, emulsions, dispersible powders or granules,syrups or elixirs), for topical use (for example as creams, ointments,gels, or aqueous or oily solutions or suspensions), for administrationby inhalation (for example as a finely divided powder or a liquidaerosol), for administration by insufflation (for example as a finelydivided powder), or for parenteral administration (for example as asterile aqueous or oily solution for intravenous, subcutaneous,intramuscular or intraperitoneal dosing or as a suppository for rectaldosing). It may be desirable to administer the therapeutic agentdirectly to or at the site of interest, e.g., injection into arthriticjoint. This may, for example, obviate any non-specific cellular sideeffects that the agent may cause.

[0232] The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

[0233] In addition to the compounds of the present invention thepharmaceutical composition of this invention may also contain, or beco-administered (simultaneously or sequentially) with, one or morepharmacological agents of value in treating one or more diseaseconditions referred to herein, for example, agents such as FK506,Cyclosporin A, steroids, azathioprine, mycophenolate mofetil,leflunomide, methotrexate and antibodies against TNF and its receptor.

[0234] The pharmaceutical compositions of this invention will normallybe administered to a warm-blooded animal at a unit dose within the range5-5000 mg per square meter body area of the animal, i.e., approximately0.1-100 mg/kg, and this normally provides a therapeutically-effectivedose. A unit dose form such as a tablet or capsule will usually contain,for example 1-250 mg of active ingredient. Preferably, a daily dose inthe range of 1-50 mg/kg is employed. In general, lower doses will beadministered when a parenteral route is employed. Thus, for intravenousadministration, a dose in the range of, for example, 0.5 mg to 30 mg perkg body weight will generally be used. Similarly, for administration byinhalation, a dose in the range of, for example, 0.5 mg to 25 mg per kgbody weight will be used. Oral administration is preferred, particularlyin tablet form. Typically, unit dosage forms will contain about 1 mg to500 mg of a compound of this invention. However, the size of the dosefor therapeutic or prophylactic purposes will naturally vary accordingto the nature and severity of the conditions, the age and sex of theanimal or patient and the route of administration, according to wellknown principles of medicine. Accordingly, the practitioner who istreating a particular patient may determine the optimal dosage. Atherapeutically effective dose or amount refers to that amount of theagent sufficient to prevent development of or to alleviate the existingsymptoms associated with the disorder. Determination of the effectiveamounts is well within the capabilities of those skilled in the art,especially in light of the detailed disclosure herein. For example, thetherapeutically effective amount can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating concentration range that includes the IC50 (concentration atwhich 50% of the maximal effect is demonstrated) as determined in invitro cellular assays. Such information can be used to more accuratelydetermine the therapeutically effective dose in humans.

[0235] The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. A formulation intended for oral administration to humanswill generally contain, for example, from 0.5 mg to 0.5 g of activeagent compounded with an appropriate and convenient amount ofexcipients, which may vary from about 5 to about 98 percent by weight ofthe total composition.

[0236] Techniques for formulation and administration of agents for usein accordance with the present invention may be found in the latestedition of “Remington's Pharmaceutical Sciences”, Mack Publishing Co.,Easton, Pa.

[0237] According to one aspect of the invention, there is provided theuse of a compound capable of inhibiting cellular monocarboxylatetransport, or a pharmaceutically-acceptable composition thereof, in themanufacture of a medicament for use in the production of ananti-proliferative effect in a warm-blooded animal, such as man. Ananti-proliferative effect is defined herein as the ability to preventcell number expansion. In one embodiment the compound is a selectiveinhibitor; in another embodiment it has broad spectrum activity.

[0238] According to a further feature of this aspect of the inventionthere is provided a method for producing an anti-proliferative effect ina warm-blooded animal, such as man, in need of such treatment, whichmethod comprises administering to said animal an effective amount of acompound capable of inhibiting monocarboxylate transport within a cell,or a pharmaceutically-acceptable composition thereof, as definedhereinbefore. Again, in one embodiment the compound is a selectiveinhibitor, while in another embodiment it has broad spectrum activity.

[0239] A variety of gene therapy approaches may be used in accordancewith the invention to modulate expression of an MCT gene in vivo. Onetherapeutic means of inhibiting or dampening the expression levels of aparticular gene (for example one of the MCT proteins) is to useantisense therapy. Antisense therapy utilises antisense nucleic acidmolecules that are synthetic segments of DNA or RNA(“oligonucleotides”), designed to mirror specific mRNA sequences andblock protein production by inhibiting translation of the native genetranscript. Once formed, the mRNA binds to a ribosome, the cell'sprotein production “factory” which effectively reads the RNA sequenceand manufactures the specific protein molecule dictated by the gene. Ifan antisense molecule is delivered to the cell (for example as nativeoligonucleotide or via a suitable antisense expression vector), it bindsto the messenger RNA because its sequence is designed to be a complementof the target sequence of bases. Once the two strands bind, the mRNA mayno longer dictate the manufacture of the encoded protein by the ribosomeand/or is rapidly broken down by the cell's enzymes (e.g., RNaseH),thereby freeing the antisense oligonucleotide to seek and disableanother identical messenger strand of mRNA. Oligonucleotides that arecomplementary to and hybridisable with any portion of MCT mRNA arecontemplated for therapeutic use. U.S. Pat. No. 5,639,595,“Identification of Novel Drugs and Reagents”, issued Jun. 17, 1997,wherein methods of identifying oligonucleotide sequences that display invivo activity are thoroughly described, is herein incorporated byreference. Expression vectors containing random oligonucleotidesequences derived from previously known polynucleotides are transformedinto cells. The cells are then assayed for a phenotype resulting fromthe desired activity of the oligonucleotide. Once cells with the desiredphenotype have been identified, the sequence of the oligonucleotidehaving the desired activity can be identified. Identification may beaccomplished by recovering the vector or by polymerase chain reaction(PCR) amplification and sequencing the region containing the insertednucleic acid material. Antisense molecules can be synthesised forantisense therapy. These antisense molecules may be DNA, stablederivatives of DNA such as phosphorothioates or methylphosphonates, RNA,stable derivatives of RNA such as 2′-O-alkylRNA, or otheroligonucleotide mimetics. U.S. Pat. No. 5,652,355, “HybridOligonucleotide Phosphorothioates”, issued Jul. 29, 1997, and U.S. Pat.No. 5,652,356, “Inverted Chimeric and Hybrid Oligonucleotides”, issuedJul. 29, 1997, which describe the synthesis and effect ofphysiologically-stable antisense molecules, are incorporated byreference. It is preferred that the sequence be at least 17 nucleotidesin length in order to achieve sufficiently strong annealing to thetarget mRNA sequence to prevent translation. Antisense oligonucleotidestargeting MCT1 (in Caco-2 cells) have been described by Hadjiagapiou etal. (Am. J. Physiol. Gastrointest. Liver Physiol. 279:G775-G780, 2000).Antisense molecules may be introduced into cells by microinjection,liposome encapsulation or by expression from vectors harboring theantisense sequence.

[0240] Alternatively, ribozyme molecules may be designed to cleave anddestroy the MCT mRNAs in vivo. Ribozymes are RNA molecules that possesshighly specific endoribonuclease activity. Hammerhead ribozymes comprisea hybridising region that is complementary in nucleotide sequence to atleast part of the target RNA, and a catalytic region that is adapted torecognise and cleave the target RNA. The hybridising region preferablycontains at least 9 nucleotides. The design, construction and use ofsuch ribozymes are well known in the art and are more fully described inHaselhoff and Gerlach (Nature 334:585-591, 1988). In anotheralternative, oligonucleotides designed to hybridise to the 5′-region ofthe MCT gene so as to form triple helix structures may be used to blockor reduce transcription of the MCT gene. In yet another alternative, RNAinterference (RNAi) oligonucleotides or short (20-25 bp) RNAi MCTsequences cloned into plasmid vectors are designed to introduce doublestranded RNA into mammalian cells to inhibit and/or result in thedegradation of MCT messenger RNA. MCT RNAi molecules may begin withadenine/adenine (AA). They may be 20, 21, 22, 23, 24 or 25 base pairdouble stranded RNA molecules with the preferred length being 21 basepairs and 2-nucleotide 3′ overhangs, alternatively they may be hairpinforming 45-50 mer RNA molecules. They would be specific for MCT mRNA.The design, construction and use of such molecules is well known in theart and is more fully described in Elbashir et al. (Nature.411(6836):428-429, 2001). In one embodiment, the antisense, ribozyme,triple helix or RNAi nucleotides are designed to specifically inhibittranslation and/or transcription of only one MCT, with minimal effectson the other MCT genes.

[0241] Thus, according to another aspect of the invention there isprovided a method for treating a patient suffering from animmune-mediated disorder or cancer (particularly cancer other than a CNScancer such as glial cell cancer), comprising identifying a patient inneed of such treatment and administering to said patient an effectiveamount of an anti-sense molecule, a ribozyme molecule, triple helixforming molecule or RNAi molecule capable of binding to the mRNA of anMCT, as hereinbefore described, including any nucleic acid or proteinderived inhibitor of transcription or translation of MCTs.

[0242] There is also provided the use of an antisense nucleic acidmolecule, a ribozyme molecule, a triple helix forming molecule, RNAimolecule or an antibody directed against an MCT, in the treatment of, ormanufacture of a medicament for treating, a cell proliferative disorder.

[0243] In the context of the present specification, the term “therapy”also includes “prophylaxis” unless there are specific indications to thecontrary. The terms “therapeutic” and “therapeutically” should beconstrued accordingly.

[0244] Prophylaxis is expected to be particularly relevant to thetreatment of persons who have suffered a previous episode of, or areotherwise considered to be at increased risk of, the disease orcondition in question. Persons at risk of developing a particulardisease or condition generally include those having a family history ofthe disease or condition, or those who have been identified by genetictesting or screening to be particularly susceptible to developing thedisease or condition.

[0245] The invention is further described, but in no way limited, by thefollowing examples:

EXAMPLE 1 Design and Construction of Ligands

[0246] 1A Ligand A

[0247] i)2,6-Dihydro-6-[(2-iodophenyl)methyl]-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0248]2,6-Dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one(205 mg), 1-chloromethyl-2-iodo-benzene (300 mg) [see WO99/29695], andcaesium carbonate (360 mg) were mixed in dry DMF (1.5 ml). Afterstirring at room temperature for 2.5 hr under nitrogen the reaction wasevaporated to dryness, and the residue was partitioned between ethylacetate and dilute HCl. The organic solution was washed with brine,dried and evaporated. The residue was purified by chromatography to givea solid, which was recrystallised from cyclohexane/ethyl acetate toafford the sub-title compound 270 mg.

[0249] MS (APCI+ve) (M+H)⁺422 NMR ¹H δ_((CDCl3)) 0.96(6H, d), 2.14(1H,m), 2.56(2H, d), 3.72(3H, s), 5.32(2H, s), 6.86(1H, d), 7.05(1H, d),7.06(1H, t), 7.33(1H, t), 7.47(1H, d), 7.90(1H, d).

[0250] ii)7-[[3-[[(1,1-Ddimethylethyl)dimethylsilyl]oxy]propyl]thio]-2,6-dihydro-6-[(2-iodophenyl)methyl]-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0251] The product of step (i) (1.1 g) andS-[3-[[(1,1-dimethylethyl)dimethylsilyl]propyloxy]-1-(4-methylphenyl)dioxidosulfanyl-propanethiol4-methyl-benzenesulfonothioate (1.7 g) were combined in THF(40 ml) undernitrogen at −78° C., and a solution of lithium diisopropylamide (0.6M,8.3 ml) was added dropwise. The reaction was quenched with saturatedaqueous sodium bicarbonate solution after 2 hr at −78° C. and then themixture was extracted into ethyl acetate. The organic solution waswashed with brine, dried and evaporated. The residue was purified bychromatography to afford the sub-title compound (400 mg).

[0252] NMR ¹H δ_((CDCl3)) 0.0(6H, s), 0.84(9H, s), 0.95(6H, d), 1.70(2H,m), 2.08(1H, m), 2.52(2H, d), 3.09(2H, t), 3.60(2H, t), 3.74(3H, s),5.47(2H, s), 6.40(1H, d), 6.99(1H, t), 7.07(1H, s), 7.25(1H, t),7.90(1H, d).

[0253] iii)7-[[3-[[(1,1-Ddimethylethyl)dimethylsilyl]oxy]propyl]thio]-2,6-dihydro-2-methyl-4-2-methylpropyl)-6-[[2-(trimethylstannyl)phenyl]methyl]-1H-pyrrolo[3,4-d]pyridazin-1-one

[0254] The product of step (ii) (34 mg) in dry toluene (1 ml) wasdegassed by purging with nitrogen, and then hexamethyl ditin (100microlitre) and tetrakistriphenylphosphine palladium (0) (10 mg) wereadded. The mixture was heated and stirred under nitrogen in a sealedflask at 95° C. for 4 hr. The reaction was cooled, diluted with ethylacetate and filtered through a pad of silica, and then purified bychromatography to afford the sub-title compound (20 mg).

[0255] NMR ¹H δ_((CDCl3)) 0.0(6H, s), 0.4(9H, s+Sn satellites at 0.32and 0.49), 0.86(9H, s), 0.96(6H, d), 1.70(2H, m), 2.08(1H, m), 2.52(2H,d), 3.06(2H, t), 3.58(2H, t), 3.75(3H, s), 5.50(2H, s), 6.54(1H, d),6.98(1H, t), 7.25-7.29(2H, m), 7.54(1H, d).

[0256] iv)2,6-Ddihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4-(2-methylpropyl)-6-[[2-(trimethylstannyl)phenyl]methyl]-1H-pyrrolo[3,4-d]pyridazin-1-one

[0257] The product of step (iii) (6 mg) was treated with a 1M solutionof TBAF in THF (0.5 ml). After 1.5 hr the reaction was diluted withsaturated aqueous sodium bicarbonate solution, and then extracted intoethyl acetate. The organic solution was washed with brine, dried andevaporated. Chromatography of the residue gave the sub-title compound (3mg).

[0258] NMR ¹H δ_((CDCl3)) 0.39(9H, s+Sn satellites at 0.33 and 0.46),0.94(6H, d), 1.73(2H, m), 2.08(1H, m), 2.52(2H, d), 3.00(2H, t),3.74(3H, s), 3.80(3H, m), 5.50(2H, s), 6.54(1H, d), 6.50(1H, d),7.01(1H, s), 7.21-7.29(2H, m), 7.54(1H, d).

[0259] v)2,6-Dihydro-7-[(3-hydroxypropyl)thio]-6-[(2-[¹²⁵I]iodophenyl)methyl]-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0260] To a solution of sodium [¹²⁵I]iodide (Amersham Pharmacia Biotech;≈2000 Ci mmol⁻¹, 1 mCi; 0.5 nmol, 10 μl) was added a solution of theproduct of step (iv) in methanol (10 μl, 3.65 nmol; 200 μg ml⁻¹, 365nmol ml⁻¹) followed by chloramine-T in water (4 μl, 8.8 nmol; 50 μgml⁻¹, 2192 nmol ml⁻¹). The vial was sealed, shaken vigorously and leftto stand at room temperature for 30 min.

[0261] The product was purified by preparative HPLC. The radiochemicalpurity was typically >98%. The radioactive concentration was determinedby liquid scintillation counting and was normally found to be in therange of 5 to 8 MBq ml⁻¹. The radiochemical yield was typically between50-60%.

[0262] vi)2,6-Dihydro-7-[(3-hydroxypropyl)thio]-6-[(2-iodophenyl)methyl]-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0263] (Cold Ligand A)

[0264] The title compound was prepared by the method of step (iv) usingthe product of step (ii).

[0265] MS (APCI+ve) (M+H)⁺512 NMR ¹H δ_((CDCl3)) 0.95(6H, d), 1.77 (2H,quintet), 2.10 (1H, septet), 2.54 (2H, d), 3.08 (2H, t), 3.74 (3H, s),3.86-3.94 (3H, m), 5.50 (2H, s), 6.37-6.41 (1H, m), 6.99-7.06 (1H, m),7.10 (1H, d), 7.22-7.28 (1H, m), 7.91 (1H, d).

[0266] 1B Ligand B

[0267] i) 1-Azido-4-chloromethyl-2-iodo-benzene

[0268] 4-Azido-3-iodo-benzenemethanol (J. Labelled Compd. Radiopharm.,1996, 38:227-37) (350 mg) in dry dichloromethane (20 ml) was treatedwith triethylamine (185 microlitre) and methanesulphonyl chloride (100microlitre) at room temperature for 20 hr. Volatiles were removed invacuo and the residue was purified by chromatography to give thesub-title compound 210 mg.

[0269] NMR ¹H δ_((CDCl3)) 4.51(2H, s), 7.11(1H, d), 7.42(1H, dd),7.82(1H, d).

[0270] ii)6-[(4-Azido-3-iodophenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one(unlabelled ligand B)

[0271]2,6-Dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one(WO99/29695) (25 mg), the product of step (i) (40 mg) and caesiumcarbonate (40 mg) were mixed in dry DMF (5 ml). After stirring at roomtemperature for 3 days under nitrogen the reaction was poured into waterand extracted into ethyl acetate. The organic solution was washed withbrine, dried and evaporated. The residue was purified by chromatographyto give the sub-title compound 50 mg.

[0272] MS (APCI+ve) (M+H)⁺463 MP 138-9° C. NMR ¹H δ_((CDCl3)) 0.97 (6H,d), 2.06-2.20 (1H, m), 2.55 (2H, d), 3.72 (3H, s), 5.20 (2H, s), 6.98(1H, d), 7.11 (1H, d), 7.17 (1H, dd), 7.47 (1H, d), 7.62 (1H, d).

[0273] iii)6-[(4-Azido-3-(trimethylstannyl)phenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0274]6-[(4-Azido-3-iodophenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one(10 mg), hexamethyl ditin (100 μl) and tetrakis triphenylphosphine Pd(0) (2 mg) were combined in dry toluene (5 ml) and heated at 100° C.under nitrogen for 4 hours. After cooling all volatiles were removed invacuo and the residue was purified by preparative thin layerchromatography (SiO₂/ethyl acetate) to afford the sub-title compound (10mg).

[0275] NMR ¹H δ_((CDCl3)) 0.3(9H, s+Sn satellites at 0.21 and 0.4, 2×d),0.96(6H, d), 2.13(1H, m), 2.55(2H, d), 3.72(3H, s), 5.22(2H, s),7.01(1H, d), 7.10-7.22(3H, m), 7.46(1H, d).

[0276] iv)6-[(4-Azido-3-[¹²⁵I]iodophenyl)methyl]-2,6-dihydro-2-methyl-4-(2-methylpropyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0277] To a solution of sodium [¹²⁵I]iodide (Amersham Pharmacia Biotech,IMS30; ≈2000 Ci mmol⁻¹, 1 mCi; 0.5 nmol, 10 μl) was added a solution ofthe product of step (iii) (10 μl, 3.0 nmol; 150 μg ml⁻¹, 300 nmol ml⁻¹)followed by chloramine-T in water (2 μl, 3.6 nmol; 500 μg ml⁻¹, 1800nmol ml⁻¹). The vial was sealed, shaken and left to stand at roomtemperature for 10 minutes. An aliquot of sodium metabisulphite (2 μl,16 nmol, 1500 μg ml⁻¹, 8000 nmol ml⁻¹) was added to the reactionfollowed by methanol (25 μl) and the vial shaken.

[0278] The iodo-azide product was purified using preparative HPLC. Theradiochemical purity was typically >99%. The radioactive concentrationwas determined by liquid scintillation counting and was normally foundto be in the range of 2 to 3 MBq ml⁻¹. The radiochemical yield wastypically between 20-30%.

[0279] 1C Ligand C

[0280] i)1,2,3,4-Tetrahydro-1-(2-hydroxypropyl)-3,6-dimethyl-2,4-dioxo-5-pyrimidinecarbonitrile

[0281] Ethyl N-(2-cyano-3-ethoxy-1-oxo-2-butenyl)-carbamate (Chem.Pharm. Bull, 1972, 20, 1380-8) (5 g) was dissolved in ethanol (50 ml) atreflux under nitrogen, and DL-1-amino-2-propanol (1.88 ml) added. After5 hr at reflux the reaction was cooled and evaporated to dryness. Theresulting gum was suspended in water (50 ml), treated with sodiumhydroxide (1.46 g) and stirred 1 hr. Dimethyl sulphate (3.45 ml) wasadded and stirring was continued for 1 hr. The precipitate wascollected, and the aqueous solution was concentrated, then extractedinto dichloromethane. The organic solution was dried and evaporated andthe residue was combined with the precipitate (above) to afford thesubtitle compound (4.35 g).

[0282] MS (EI) (M+H)⁺223 BP 159

[0283] ii)1-(2-Hhydroxypropyl)-3-methyl-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione

[0284] The product of step (i) (4.24 g) was suspended in 75% formic acid(80 ml) and Raney Nickel (50% dispersion in 8 ml water) was added. Themixture was heated at 90° C. under nitrogen for 15 min. After coolingthe suspension was filtered (kieselguhr) and evaporated. The residue wasdissolved in water (100 ml) and extracted into ethyl acetate, eachaliquot of extraction was washed with sodium bicarbonate solution. Thecombined organic extracts were dried and evaporated to yield a whitefoam, which was dissolved in chloroform (20 ml) and heated to 50° C. Asolution of bromine (0.4 ml) in chloroform (5 ml) was added and afterstirring for 10 min at 50° C. was concentrated in vacuo. The residue wasdissolved in ethanol (25 ml), treated with triethylamine (2.96 ml) andthen 1-naphthalenylmethylamine (1.55 ml) was added. After 20 hr at roomtemperature the reaction was poured into 2M HCl (100 ml) and extractedwith ethyl acetate, dried (MgSO₄) and then concentrated in vacuo.Purification by chromatography (SiO2/2:1 hexane-ethyl acetate) gave thesub-title compound, which was crystallised from hexane/ethyl acetate toafford sub-title compound, 110 mg.

[0285] MS (EI) (M+H)⁺ 363 BP 141

[0286] iii )3-Methyl-6-(1-naphthalenylmethyl)-1-(2-oxopropyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione

[0287] A solution of anhydrous DMSO (417 μl) in anhydrousdichloromethane (10 ml) was added dropwise to a solution of oxalylchloride (256 μl) in dichloromethane (20 ml) at −78° C. under nitrogen.After 15 min a solution of the product of step (ii) (970 mg) indichloromethane (20 ml) at −78° C. was added. After 5 min triethylamine(900 μl) was added, the reaction was stirred 10 min then allowed to warmto 0° C. Water (100 ml) was added and the mixture was extracted withdichloromethane. Drying (MgSO₄), evaporation and chromatography gave thesub-title compound (660 mg).

[0288] MS (EI) (M+H)⁺ 361 BP 141

[0289] iv)3-Methyl-1-(2-methyl-2-propenyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione

[0290] A stirred suspension of methylene triphenylphosphonium bromide(1.22 g) in dry THF (20 ml) at −78° C. under nitrogen was treated withsodium hexamethyldisilazide (3.1 ml of 1M solution in THF). The reactionmixture was stirred at room temperature for 1 hr. The resulting solutionwas added to a solution of the product of step (iii) (560 mg) in dry THF(30 ml) at 0° C. under nitrogen, and stirred at 5° C. for 2 hr then 20min at room temperature. The mixture was poured into water (50 ml) andextracted into ethyl acetate. Drying, evaporation and chromatographygave the sub-title compound (465 mg).

[0291] MS (EI) (M+H)⁺ 339 BP 141

[0292] v)5-[(3-Hydroxypropyl)thio]-3-methyl-1-(2-methyl-2-propenyl)-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione

[0293] The product of step (iv) (350 mg) andS-[3-[[(1,1-dimethylethyl)dimethylsilyl]propyloxy]-1-(4-methylphenyl)dioxidosulfanyl-propanethiol4-methyl-benzenesulfonothioate (527 mg) were dissolved in dry THF (10ml) at −78° C. under nitrogen. LDA (1.95 mmol) in dry THF (5 ml) wasadded, and after 1 hr the temperature was raised to 0° C. The reactionwas quenched by addition of sodium bicarbonate solution (30 ml), andextracted into ether. Drying and evaporation gave a residue which wasdissolved in acetonitrile (10 ml) and treated with 40% HF (0.4 ml) for30 min. The reaction mixture was poured into sodium bicarbonate solutionand extracted into ethyl acetate. Drying, evaporation and chromatographygave the sub-title compound which was recrystallised from hexane-ethylacetate afford the product (163 mg).

[0294] NMR ¹H nmr δ_((CDCl3)) 1.67(3H, s), 1.8(2h, m), 3.1(2H, t),3.44(3H, s), 3.83(2H, dd), 4.53(2H, s), 4.72(1H, s), 4.83(1H, s),5.83(2H, s), 6.37(1H, s), 6.76(1H, d), 7.39(1H, t), 7.58(2H, m),7.83(1H, d), 7.79(2H, m).

[0295] vi)5-[(3-Hydroxypropyl)thio]-1-([2,3,3′-³H]isobutyl)-3-methyl-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione5-[(3-Hydroxypropyl)thio]-3-methyl-1-[2,3-di³H-2-(³H-methyl)propyl]-6-(1-naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione

[0296] The product of step (v) (2.28 mg, 5.1 μmol), 10% Pd/carbon (2.35mg) and ethanol (0.5 ml) were placed in a 1 ml round-bottomed flaskwhich was attached to a tritium manifold. The contents of the flask werefrozen in liquid nitrogen and the flask then evacuated before tritiumgas (241 GBq, 2.6 ml, 0.113 mmol) was introduced. The flask was allowedto warm to room temperature and the contents left to stir for 22 hours.

[0297] The flask was removed from the apparatus and the catalyst removedby filtration. The filtrate was diluted with ethanol (5 ml) and thesolvent removed under reduced pressure. This was repeated with a furtherportion of ethanol (5 ml).

[0298] Purification of the tritiated material was achieved byreversed-phase HPLC using a Waters Novapak C₁₈ 200×8 mm radialcompression module eluting with 45% v/v acetonitrile/0.1% v/v aqueoustrifluoroacetic acid at 3 ml min⁻¹ and UV detection at 254 nm. Analiquot of the stock solution (5 ml) was reduced to dryness andre-dissolved in acetonitrile (0.5 ml) and purified in approximatelythree equal injections. The peak due to ligand C was collected as threeseparate fractions (front, middle and back) and these were combined withthe equivalent cuts from the two subsequent injections. The volumes ofthe three fractions were measured in each case and made up to 10 ml bythe addition of 50% w/v aqueous sodium thiosulphate (100 μl) andethanol. The radioactive concentration, molar specific activity andradiochemical purity of the three fractions was determined and thedetails included in the table below. TABLE 3 Radioactive Molar SpecificRadio- Concentration Activity chemical Fraction (MBq ml⁻¹) (GBq mmol⁻¹)Purity Front fraction 24.53 1713.1 97.8% Middle fraction 34.78 1631.797.9% Back fraction 14.39 1217.3 ≈78%

[0299] Both the front and middle fractions provided material of asuitable radiochemical purity and specific activity for use in theligand binding assay.

[0300] Unlabelled ligand C is disclosed in Michne et al. (J Med. Chem.(1995) 38:2557-2569).

EXAMPLE 2 Photoaffinity Labelling of the Target Protein

[0301] The unlabelled compounds and radioligands of Example 1 were usedto identify the precise target(s) involved in the binding interactionusing photoaffinity labelling, gel electrophoresis, peptide sequencingand mass spectrometry. The target protein was identified as being MCT1.

[0302] Photoaffinity labelling was performed using ¹²⁵I-labelled ligandB. Photolabelling reactions were set up by diluting washed rat red-bloodcell membranes 10-fold in assay buffer (50 mM HEPES (pH 7.5); 0.1 mMEDTA; 150 mM NaCl) and by incubating in the presence or absence of 1 μMunlabelled competing ligand C. The ¹²⁵I-photoligand solution was addedto give a final ligand concentration of 1 nM. The photolabellingreaction was performed by irradiating the sample with a hand held 254 nmUV source for 1 minute at room temperature. The labelled ghost membraneswere then collected by centrifugation at 100,000 g for 10 minutes at 4°C. Finally, the samples were each washed in 1 ml of distilled water andthe final membrane pellets were collected at 100,000 g for 10 minutes at4° C. The samples were stored at −20° C. until use.

[0303] The photoaffinity-labelled proteins were analysed by one- andtwo-dimensional gel electrophoresis. The labelled proteins were excisedfrom the gel and subjected to in gel protein digestion prior to analysesby mass spectrometry. Three peptides matching rat monocarboxylatetransporter 1 were identified.

EXAMPLE 3 Cloning of MCT Genes

[0304] 3.1 Human MCT1

[0305] Oligonucleotide primers containing unique restriction sites, toallow subsequent cloning, and sequences derived from the optimal Kozakconsensus sequence (MCT1-5′;5′-GGA-TCC-ACC-ATG-CCA-CCA-GCA-GTT-GGA-GG-3′; SEQ ID No: 41; andMCT1-3′; 5′-GTC-GAC-TCA-GAC-TGG-ACT-TTC-CTC-CTC-CTT-G-3′; SEQ ID No: 42)were used in a PCR to amplify the MCT1 ORF from a cDNA library. The PCRfragment was subcloned into the vector pCR3.1 uni (Invitrogen).Bacterial colonies containing the MCT1 ORF in the vector were identifiedin a PCR colony screen. A number of MCT1 positive colonies were grown,the plasmid DNA isolated and subjected to sequence analysis to ensurethat no amino acid encoding mutations had been incorporated into theMCT1 ORF. The MCT1 ORF (BamHI/SalI) was then sub-cloned into themammalian expression vector pcDNA3 (Invitrogen) and digested withBamHI/XhoI to generate the plasmid pcDNA3-hMCT1. The MCT1 ORF was thenfurther subcloned into the insect expression vector pIZv5HIS(Invitrogen) and the S. cerevisiae expression plasmid, pACES14.Expression plasmids pcDNA3-hMCT1, pIZ-hMCT1, and pACES14-hMCT1 werepurified and used to transform relevant host cells.

[0306] 3.2 Rat MCT1

[0307] The open reading frame encoding rat MCT1 was amplified from a ratbrain cDNA library (Origene) using the olIgonucleotide pair RM1-5′(5′-TGCATGATCA-ATGCCACCTGCGATTGGCGGGCCAG-3′; SEQ ID No. 8) and RM1-3′(5′-TGCAGCTAGCTCAG-ACTGGGCTCTCCTCCT-3′; SEQ ID No. 9) in a PCR. Theresulting amplified DNA was digested with BclI/NheI and was ligated withpACES14 pre-digested with BamHI/NheI. The resulting insert DNA wassequenced to ensure that no mutations had been incorporated. PlasmidpACES14-rMCT1 was purified and used to transform relevant host cells.

[0308] 3.3 Human MCT2

[0309] The open reading frame encoding human MCT2 was amplified from afull-length cDNA clone using the oligonucleotide pair MCT2-5′(5′-AGC-TGG-ATC-CAC-CAT-GCC-ACC-AAT-GCC-AAG-3′; SEQ ID No. 10) andMCT2-3′ (5-GAC-TCT-CGA-GTT-AAA-TGT-TAG-TTT-CTC-TTT-CTG-A-3′; SEQ ID No.11) in a PCR. The PCR fragment was subcloned into the vector pCR3.1 uni(Invitrogen). Bacterial colonies containing the MCT2 ORF in the vectorwere identified in a PCR colony screen. A number of MCT2 positivecolonies were grown and the plasmid DNA isolated and subjected tosequence analysis to ensure that no amino acid-altering mutations hadbeen incorporated into the MCT2 ORF. The full length open reading framefor human MCT2 was then subcloned into the S. cerevisiae expressionplasmid, pACES14, the insect expression vector pIZv5HIS (Invitrogen),and the mammalian expression vector pcDNA3 (Invitrogen). PlasmidspACES14-hMCT2, pIZ-hMCT2, and pcDNA3-MCT2 were purified and used totransform relevant host cells.

[0310] 3.4 Human MCT3

[0311] The human MCT3 ORF spans 4 exons in the human genome (Yoon etal., Genomics 60 (3), 366-370, 1999). Each of the four exons wasamplified by PCR from human genomic DNA using the oligosMCT3-5′#3(5′-ATC-AGG-ATC-CAG-GCA-GCG-ATG-GGC-G-3′; SEQ ID No.12)/MCT3-11# (5′-GAC-ACG-GGG-CCC-GTG-CCG-TAG-AGC-AT-3′; SEQ ID No. 13)for exon I; MCT3-10#(5′-CGG-CAC-GGG-CCC-CGT-GTC-CAG-CAT-3′; SEQ IDNo.14)/MCT3-13# (5′-AGG-CCC-AGG-CCT-GTG-AGC-ACC-CCA-GC-3′; SEQ ID No.15) for exon II; M3-G1(5′-GTT-CCC-GGA-TCT-GCT-GGG-TT-3′; SEQ ID No.16)/M3-G2 (5′-TGG-AGC-TTC-CCT-GGG-TCT-AA-3′; SEQ ID No. 17) for exon IIIflanked by intron DNA; MCT3-14#(5′-CCC-TCT-GCC-GGC-CGC-CTG-GTG-GAT-GCG-TTG-AAG-3′; SEQ ID No.18)/MCT3-3′#3 (5′-GTC-AAC-TAG-TCA-GAC-ACC-CAG-GGG-ATC-AAC-TGG-AG-3′; SEQID No. 19) for exon IV to ˜150 bp downstream of the termination codon.Exon III was then isolated the M3-G1/M3-G2 PCR product using the oligosMCT3-12# (5′-TGC-TCA-CAG-GCC-TGG-GCC-TGG-CCC-TCA-A-3′; SEQ ID No.20)/MCT3-15# (5′-ACC-AGG-CGG-CCG-GCA-GAG-GGC-GGT-CC-3′; SEQ ID No. 21).A PCR product (I+II) was generated from the exon I and exon II PCRproducts using the oligos MCT3-5′#3/MCT3-13# and subcloned intopCRBluntII-TOPO to give pTOPOMCT3(I+II). A PCR product (III+IV) wasgenerated from the exon III and exon IV PCR products using the oligosMCT3-12#/MCT3-3′#3 and subcloned into pCRII-TOPO to givepTOPOMCT3(III+IV). The MCT3 fragments from HindIII/StuI digestedpTOPOMCT3(I+II) and from HindIII/StuI digested pTOPOMCT3(III+IV) wereligated to give the full length pTOPOMCT3 (in the pCRBluntII vector). Anumber of MCT3 positive colonies were grown and the plasmid DNA isolatedand subjected to sequence analysis to ensure that no amino acid encodingmutations had been incorporated into the MCT1 ORF. Following sequenceanalysis, the full length open reading frame of human MCT3 was subclonedinto the S. cerevisiae expression plasmid pACES14, the insect expressionvector pIZv5HIS (Invitrogen), and the mammalian expression vector pcDNA3(Invitrogen). Plasmids pACES14-hMCT3, pIZ-hMCT3 and pcDNA3-hMCT3 werepurified and used to transform relevant host cells.

[0312] 3.5 Human MCT4

[0313] A human dendritic cell cDNA clone (AC-DNA-1819) contains anincomplete copy of the MCT4 open reading frame. This was made fulllength by inserting ˜190 bp GBO212(5′-TAG-GAA-GAA-GCC-CAA-AGA-GCC-ACA-G-3′; SEQ ID No. 22)/GBO213(5′-GAC-TTC-TAG-AGC-CCA-GCC-ACT-CAG-ACA-CTT-GTT-TC-3′; SEQ ID No. 23)MCT4 3′ PCR fragment (amplified from a human naive T cell cDNA stock) ona NotI—XbaI fragment to make pGBAC41. The MCT4 ORF was subsequentlyamplified (using oligonucleotides GBO214(5′-GAT-CGG-ATC-CAT-GGG-AGG-GGC-CGT-GGT-3′; SEQ ID No. 4)/GBO215(5′-GTC-AGA-TAT-CGC-CAC-TCA-GAC-ACT-TG-3′; SEQ ID No. 25), which addBamHI and EcoRV restriction site recognition sequences to the 5′ and 3′ends, respectively) from the full length copy and subcloned into the S.cerevisiae expression plasmid pACES14. The insert was sequenced toensure that no mutations that would result in incorporation of alteredamino acids had been incorporated during amplification and cloning. Thehuman MCT4 ORF was then inserted into the insect expression vectorpIZv5HIS (Invitrogen) and the mammalian expression vector pcDNA3.Plasmids pACES14-hMCT4, pIZ-hMCT4, and pcDNA3-hMCT4 were purified andused to transform relevant host cells.

[0314] 3.6 Human MCT1/MCT2 Chimera

[0315] The human MCT1 and MCT2 cDNAs share a common recognition site forthe restriction enzyme Hind III. This restriction site lies in theregion that encodes the extracellular domain that separates thepredicted transmembrane domains 5 and 6. The common Hind III site wasused to create a human MCT1/MCT2 chimeric molecule, in the S. cervisiaeexpression plasmid pACES14, consisting of the amino terminus of MCT2(TMs 1-5) and the carboxy terminus of MCT1 (TMs 6-12) (SEQ ID No: 40).Plasmid pACES14-hMCT2/MCT1 was purified and used to transform relevanthost cells.

[0316] A series of further chimeric molecules was constructed byreplacing parts of MCT1 with MCT2. Each of these bound ligand in thelatter binding studies to greater or lesser extent.

EXAMPLE 4 Expression of MCT Proteins in Host Cells

[0317] The human breast cell line MDA-MB-231 had been previouslyidentified as expressing low levels of MCT1 (Garcia et al., Cell.76:865-873, 1994). The MDA-MB-231 cell line was grown. Filter bindingassays (see Examples 6 and 7) carried out with the MDA-MB-231 cell lineshowed that the cells exhibited low level of binding to ³H-ligand C(˜6000 binding sites per cell). The cells were grown and transfectedwith pcDNA3-hMCT1. An increase in filter binding of ³H-ligand C wasconsistently measured for transiently transfected cells when comparedwith control cells transfected with empty vector.

[0318] The rat pancreatic β-cell line INS-1 has also been shown toexhibit low levels of lactate transport activity (Sekine et al., J.Biol. Chem. 269:4895-4902, 1994) and has been shown to express lowlevels of MCT proteins (Ishihara et al., J Clinical Invest.104:1621-1629,1999; Zhao et al., Diabetes 50:361-366, 2001). INS-1 cellswere grown and transfected with pcDNA3-hMCT1. An increase in filterbinding of ³H-ligand C was consistently measured for transfected cellswhen compared with control cells transfected with empty vector.

[0319] Filter binding assays carried out with the insect cell line Sf9showed that the cells exhibited low level of binding to ³H-ligand C. TheSf9 cell line was grown and transfected with pIZ-hMCT1. An increase infilter binding of ³H-ligand C was consistently measured for transfectedcells when compared with control cells transfected with empty vector.

[0320] The yeast strain used for expression of MCTs was Saccharomycescerevisiae Hansen BY4742 (Research Genetics) Mat alpha his3D1 leu2D0lys2D0 ura3D0 Δjen:Kan^(r). Yeast cells were made competent for DNAtransformation and transformed with plasmid DNA using the YeastTransformation Kit (SIGMA) according to the manufacturer's instructions.Expression of human MCTs was confirmed by Western analysis using thecorresponding anti-human MCT C-terminal peptide antibody.

[0321] Antibody Generation:

[0322] Anti-human MCT1-4 C-terminal peptide antibodies were made byCambridge Research Biochemicals. Peptide sequences used to immuniserabbits are as follows: TABLE 4 Human MCT Peptide Sequence SEQ ID No.MCT1 CQKDTEGGPKEEESPV 26 MCT2 CKVSNAQSVTSERETNI 27 MCT3CTEPEIEARPRLAAESV 28 MCT4 CEPEKNGEVVHTPETSV 29

[0323] Three single amino acid variants of human MCT1 (Lys240Glu,Cys400Gly, Glu490Asp) were expressed in yeast cells, and membranepreparations containing these three variants were tested in filterbinding assays. No alteration to compound binding was detected whencompared to human MCT1 expressed in yeast. Single nucleotide changesthat would result in the desired amino acid changes were incorporatedinto pACES-hMCT1 using the QuickChange Site-Directed Mutagenesis Kit(Stratagene). Incorporation of a mutated nucleotide residue that wouldresult in an altered amino acid was confirmed by sequence analysis.

EXAMPLE 5 Cloning of a Strep Tag to the C-Terminus of MCT Proteins

[0324] The DNA sequence encoding the Strep Tag, AWRHPQFGG (SEQ ID No.30) (Schmidt and Skera, 1993, Protein Engineering 6:109-122), was clonedand expressed at the C-terminus of hMCT1, hMCT2 and hMCT3.

[0325] MCT1

[0326] A three way ligation containing pACES14 (BamHI/NheI), the 5′ endof hMCT1 (BamHI/BspE1 digested pACES14-MCT1) and annealedoligonucleotides M1strp-1(5′-CCG-GAC-CAG-AAA-GAC-ACA-GAA-GGA-GGG-CCC-AAG-GAG-GAG-GAA-AGT-CCA-GTC-GCT-TGG-AGA-CAT-CCA-CAA-TTT-GGT-GGT-TAA-T-3′;SEQ ID No. 31) and M1strp-2(5′-CTA-GAT-TAA-CCA-CCA-AAT-TGT-GGA-TGT-CTC-CAA-GCG-ACT-GGA-CTT-TCC-TCC-TCC-TTG-GGC-CCT-CCT-TCT-GTG-TCT-TTC-TGG-T-3′;SEQ ID No. 32) encoding the C-terminus of MCT1 fused to the DNA encodingthe strep-tag was carried out to generate the plasmid pACES14-MCT1-streptag. Expression of the tagged material was confirmed by Western blottingusing anti-hMCT1 Abs and a streptavidin-horseradish peroxidase conjugate(IBA GmbH). Ability of a yeast membrane preparation containing the streptagged MCT1 to bind various radioligands (³H-ligand C, ¹²⁵I-ligand A)was tested and confirmed in the filter binding assay. Yeast membranepreparations containing the strep tagged MCT1 were then used to defineconditions for an 384-well SPA assay using radioligand (¹²⁵I-ligand A)and streptavidin-coated PVT SPA beads (Amersham Pharmacia Biotech), asdescribed below.

[0327] MCT2

[0328] A three way ligation containing pACES14 (BamHI/NheI), the 5′ endof hMCT2 (BamHI/DraIII digested pIZ-MCT2) and annealed oligonucleotidesFM2strep2(5′-GTG-TAA-CCT-CAG-AAA-GAG-AAA-CTA-ACA-TTG-CTT-GGA-GAC-ATC-CAC-AAT-TTG-GTG-GTT-AAT-3′;SEQ ID No. 33) and RM2strep2(5′-CTA-GAT-TAA-CCA-CCA-AAT-TGT-GGA-TGT-CTC-CAA-GCA-ATG-TTA-GTT-TCT-CTT-TCT-GAG-GTT-ACACTCT-3′;SEQ ID No. 34) encoding the C-terminus of MCT2 fused to the DNA encodingthe strep-tag, was carried out to generate the plasmidpACES14-MCT2-strep tag. Expression of the tagged material was monitoredby Western blotting using anti-hMCT2 Abs and a streptavidin-horseradishperoxidase conjugate (IBA GmbH).

[0329] MCT3

[0330] The 3′ end of hMCT3 (from Topo-MCT3) was amplified in a PCRreaction with the primer pair Mct3E-2 (5′-GCCATCCTGCTGGTGAACTA-3′; SEQID No. 35) and M3-3st(5′-TAG-CTA-GTC-TAG-ATT-AAC-CAC-CAA-ATT-GTG-GAT-GTC-TCC-AAG-CTA-CAG-ACT-CGG-CAG-CCA-GCC-TCG-GCC-TCG-CC-3′;SEQ ID No. 36). The PCR fragment contains the 3′ end of hMCT3 fused tothe DNA encoding the strep tag. The PCR product was digested with NotIand XbaI and placed in a three-way ligation with pACES14 BamHI/NheI andthe 5′ end of hMCT3 (released from Topo-MCT3 digested with BamHI/NotI),to generate pACES14-hMCT3strep tag. Expression of the tagged hMCT3 wasmonitored by Western blotting using anti-hMCT3 Abs and astreptavidin-horseradish peroxidase conjugate (IBA GmbH). Ability of ayeast membrane preparation containing the strep tagged MCT3 to bindvarious radioligands (³H-ligand C, ¹²⁵I-ligand A) was tested in thefilter binding assay.

EXAMPLE 6 Yeast Expressed MCT1 Filter Binding Assay

[0331] Assay to Measure the Potency of Selected Compounds Using a FilterBinding Assay

[0332] Competition assays can be used to measure the affinity ofunlabelled compound for MCT1. Tritiated ligand C is included at aconstant concentration and the compound to be tested is titrated. 10 μlof ³H-ligand C that had been diluted with assay buffer (50 mM HEPES, 0.1mM EDTA, 0.15 M NaCl, pH 7.5, 0.5% BSA) was dispensed into wells of apolypropylene plate such that when the assay was made to 200 μl thefinal concentration would be 2.5 nM. 10 μl of compound in assay bufferwas added to each well to give a final concentration typically coveringthe range 0.1 to 1000 nM. 180 μl of yeast membranes expressing MCT1 inassay buffer containing typically 0.5 to 1 μg total protein was added tostart the reaction. Non-specific binding was measured in the presence of1 μM unlabelled Ligand 1. The competition assay was incubated at 2 hoursat room temperature with shaking. Experimental data points were usuallycarried out in triplicate. The membranes were harvested onto GF-B filterplates and washed with assay buffer without BSA, dried, scintillantadded and counts detected using a tritium program on a Packard Top Countplate reader. The results were analysed by subtraction of thenon-specific binding from each of the experimental points and thenfitting a sigmoidal curve through a semi-log plot of the data inMicrocal Origin. Calculated IC₅₀'s are shown in Table 5. TABLE 5Compound IC50s calculated from the yeast membrane MCT1 filter bindingassay. Ligand IC₅₀ (nM) 1 0.5 2 0.3 3 105 4 787

[0333] Key:

[0334] Ligand 1: (disclosed in WO 98/054190)5-[(3-hydroxypropyl)thio]-3-methyl-1-(2-methylpropyl)-6-(1-napthalenylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

[0335] Ligand 1 appears in patent WO 98/054190 and has CAS number (ChemAbs Registry No) 216685-07-3.

[0336] Ligand 2: (disclosed in WO 99/029695)2,6-dihydro-7-[(3-hydroxypropyl)thio]-2-methyl-4-(2-methylpropyl)-6-(1-napthanlenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one

[0337] Ligand 2 appears in patent WO 99/29695 and has CAS number227321-12-2.

[0338] Ligand 3: (disclosed in WO 98/054190)6-(4-quinolinylmethyl)--3-methyl-1-(2-methylpropyl)-thieno[2,3-d]pyrimidin-2,4(1H,3H)-dione

[0339] Ligand 4: (disclosed in WO 00/12514):6-([benzothiazol-2-yl]methyl)-3-methyl-1-(2-methylpropyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

EXAMPLE 7 MCT Filter Binding Assay

[0340] For yeast cells transformed with expression plasmids containingthe human MCT 1-4 ORFs the ability of membrane preparations to bindradioligand was determined using a single ligand concentration/multiprotein concentration binding assay. This generated an estimate of theamount of active binding protein present in each membrane preparation.The ligand concentration used will give an estimate of B_(max) that isapproximately 90% of the true value. 10 μl of ligand A, B or C that hadbeen diluted 50 fold with assay buffer (50 mM HEPES, 0.1 mM EDTA, 0.15 MNaCl, pH 7.5, 0.5% BSA) was dispensed into wells of a polypropylenemicrotitre plate. This gives a final radiolabelled ligand concentrationof ˜2.5 nM. Non-specfic binding was measured in the presence of 1 μMLigand 1. The membranes were diluted to give a known amount/180 μl assaybuffer. This is typically in the 2000 nl to 0.2 nl range. For themembranes with high binding site number a more dilute membranepreparation should be added. 180 μl of each membrane solution was thenadded to each well and incubated at 2 hours at room temperature withshaking. The membranes were harvested onto GF-B filter plates and washedwith assay buffer without BSA, dried, scintillant added and countsdetected using the relevant program for each ligand on a Packard TopCount plate reader. Results from a typical experiment are shown in Table6. TABLE 6 MCT (protein Specific binding (cpm) quantity Ligand C LigandA assayed) Average Average MCT1 (20 ug) 5553 177453 MCT1 (2 ug) 362188841 MCT2 (20 ug) 213 14769 MCT2 (2 ug) 593 7286 MCT3 (20 ug) 40 0 MCT3(2 ug) 0 0 MCT4 (20 ug) 0 872 MCT4 (2 ug) 81 405 vector (20 ug) 0 0vector (2 ug) 0 1735

[0341] The filter binding assays of Examples 6 and 7 are suitable forhigh throughput screening of large compound libraries.

EXAMPLE 8 Yeast Expressed MCT1 Based SPA Assay

[0342] Scintillation Proximity Assay Method Final conditions: 15 μgbeads/well 3 μg yeast membranes/well 0.1 nM ¹²⁵I ligand A 50 mM HEPES pH7.5, 0.1 mM EDTA, 150 mM NaCl + 0.05% BSA

[0343] Streptavidin coated SPA beads were resuspended at 2.5 mg/ml inassay buffer (50 mM HEPES pH 7.5, 0.1 mM EDTA, 150 mM NaCl) and dilutedto give a final concentration of 187 μg/ml in assay buffer without BSA.Yeast membranes expressing MCT1 with a streptavidin-binding sequence tagwere then added to give a final concentration of 37 μg/ml protein andgently rolled for >30 minutes at room temperature. The beads/membraneswere then washed by centrifugation at ˜650 g for 10 minutes andresuspended in assay buffer+0.05% BSA. The washing was repeated oncebefore resuspended in the appropriate volume of assay buffer+0.05% BSA.

[0344] The iodinated ligand A was diluted from the stock into assaybuffer+0.5% BSA to give a concentration of 1 nM. Non-specific bindingwas measured in the presence of Ligand 1 at 1 μM. Compound IC₅₀s weremeasured in triplicate over a 100,000-fold dilution range withindividual dilutions at half log units. 10 μl of diluted ligand wasadded to each well, 10 μl of cold competitor ligand 1 was added to thenon-specific control wells and 10 μl of buffer was added to the totalcounts control wells. Finally, 80 μl of the membrane/bead suspension wasadded to each well. The plates were sealed, incubated at roomtemperature for 3 hours, centrifuged at 650 g for 5 minutes beforecounting in a Packard Top Count plate reader using a protocolappropriate for ¹²⁵I. The raw counts were analysed by averagingreplicates, subtraction of non-specific binding and the subsequentcalculation of % inhibition of binding of the iodinated ligand. IC₅₀'swere calculated using Origin data fitting software and are shown inTable 7. TABLE 7 Compound IC50s calculated from the yeast membraneMCT1-strep tagged SPA assay. Ligand IC₅₀(nM) 1 0.6 2 0.5 3 15.4 4 32.2

[0345] This SPA assay is suitable for high throughput screening of largecompound libraries.

EXAMPLE 9 Uptake of Labelled Lactate in Rat Red Blood Cells

[0346] Ligand 1 was dissolved at a concentration of 10 mM in DMSO anddiluted in assay buffer (50 mM HEPES (pH 7.5), 1 mM EDTA, 50 mM NaCl)supplemented with 0.5% (w/v) BSA. 100× stock solutions were made foreach concentration and 2 μl of these stocks diluted into 200 μl of ratblood to give the final compound concentration. The blood samples wereincubated at room temperature for 2 hours. Lactate uptake was measuredin each blood sample as follows: uptake was initiated by the addition of50 μl of blood to 2 μl of ¹⁴C-lactate (12.5 μCi/ml; Amersham). Thesamples were incubated at room temperature for 30 s and then thereaction was halted by transfer of 20 μl of each sample onto 1 ml ofice-cold dibutyl-pthalate (Sigma). The red blood cells were separatedfrom the plasma by centrifugation of the samples for 30 s at 15000 g ina bench-top microfuge. The supernatant was aspirated to waste takingcare not to disturb the cell pellet. The cells were resuspended in 100μl of 5% NP40 (v/v) in PBS and were transferred to a scintillation vial.4 ml of scintillant were added and radioactivity was determined byscintillation counting. MCT1 inhibitors, such as Ligand 1, caused adose-dependent decrease in the amount of [¹⁴C]-lactate uptake by rat redblood cells. Values from representative experiments were: TABLE 8[¹⁴C]-Lactate uptake (cpm) Experiment 1 Experiment 2 Experiment 3 NoLigand 1 23000 ± 3000 22957 ± 1664 21497 ± 5304 1 μM Ligand 1 4362 ± 40910426 ± 243  11146 ± 817 

[0347] This method was also used to determine the effect of MCT1inhibitors, including Ligand 1, on lactate uptake in human red bloodcells and similar results were obtained.

EXAMPLE 10 Scintillation Proximity Assay (SPA) Using Jurkat T-CellMembranes

[0348] Human Jurkat T cells were grown at 37° C. in RPMI 1640 mediumsupplemented with 5% foetal calf serum, 2 mM glutamine. Cells wereharvested by centrifugation at 1500 g for 10 minutes. The cell pelletswere washed twice with phosphate buffered saline (PBS) and centrifugedas above. The final cell pellet was resuspended in lysis buffer (50 mMHEPES (pH 7.8), 50 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT) andlysed by nitrogen cavitation. Unlysed cells and nuclei were removed bycentrifugation at 1500 g for 10 minutes. The supernatant was thencentrifuged at 100,000 g for 30 minutes at 4° C., and the pelletresuspended and homogenised in assay buffer (50 mM HEPES (pH 7.5), 1 mMEDTA, 150 mM NaCl). Aliquots of membrane preparation were stored at −80°C. until use.

[0349] Frozen Jurkat cell membranes were thawed on ice and thenhomogenised. Wheatgerm agglutinin-linked Scintillation Proximity Assaybeads (Amersham) were rehydrated in assay buffer to a concentration of100 mg/ml. 2 ml of Jurkat membranes were added per 0.5 ml (50 mg) of SPAbeads and incubated overnight with constant agitation to allow the beadsto coat with Jurkat membranes. The coated beads were then collected bycentrifugation at 1500 g for 5 minutes and were washed twice in largevolumes of assay buffer before being resuspended to a finalconcentration of 10 mg/ml in assay buffer. ¹²⁵I-ligand A was prepared asdescribed above at a maximum specific activity of 2000 Ci mmol⁻¹. Theradioligand was diluted in assay buffer containing 0.5% (w/v) BSA; thefinal concentration in the assay was approximately 0.1 nM. Assays wereset up in 96-well flat-bottomed white opaque plates (Costar. Cat No:3912). 10 μl of test compound and 10 μl of radioligand were incubatedwith 180 μl of SPA beads and membranes (0.04 mg beads). Non-specificbinding was determined in the presence of 1 μM Ligand 1 and totalbinding was determined in the presence of vehicle alone. The plates wereincubated for 3 hours at room temperature before quantitation ofradioactivity proximal to the SPA beads by scintillation counting.

[0350] Compounds including Ligand 1 caused a dose-dependent reduction inthe specific binding of ¹²⁵I-ligand A to Jurkat T-cell membranes. Valuesfrom a representative experiment were: TABLE 9 ¹²⁵I-ligand A binding(cpm) Total binding 2490 ± 32 Non-specific binding  499 ± 20 Specificbinding (Total - Non-specific) 1991

[0351] The mean Ki for Ligand 1 competition of ¹²⁵I-ligand A binding toJurkat T-cell membranes was 0.074 nM (n=100).

EXAMPLE 11 Effect of MCT1 Inhibitors on T-Lymphocytes

[0352] Our studies have shown that the rate of lactate production byT-lymphocytes increases approximately 15-fold by 48 h after mitogenicstimulation (PMA/ionomycin). Western blotting analyses of stimulatedPBMCs using Abs that recognise MCT1, MCT2 and MCT4 showed that thesethree MCTs are expressed 48 hours after stimulation. Compounds that bindto MCT1 with potencies in the region of 0.05-300 nM have been shown tocause a significant accumulation of intracellular lactate in theT-lymphocytes and a reduction in the amount of lactate in theextracellular medium. The potency of compound effects on lactateaccumulation shows a significant correlation with inhibition ofT-lymphocyte proliferation as measured by the rate of incorporation of[³H]thymidine into DNA. Data from compound activity in an SPA bindingassay, 3-day lymphocyte proliferation assay and experiments onT-lymphocytes after 48 h of mitogenic stimulation (lactate levels andDNA synthesis) are shown.

[0353] Assay of T-Lymphocyte Proliferation

[0354] The cell signalling pathways triggered by T-cell activationthrough the T cell receptor and CD28 can be mimicked in vitro using aphorbol ester, phorbol 12-myristate 13-acetate (PMA), which activatesprotein kinase C, and ionomycin, which induces calcium release frominternal cellular stores. PMA and ionomycin-stimulated proliferation ofperipheral blood mononuclear cells (PBMC), of which the predominant celltype is T-lymphocytes, provides a suitable assay for measuring theability of small molecules to block T-cell proliferation.

[0355] 100 ml blood was collected by venopuncture of normal humanvolunteers into 3 tubes each containing 3 ml of 3.2% tri-sodium citratesolution. Blood was centrifuged at 850 g for 10 minutes and the plasmawas removed. The cells were diluted to 50 ml with RPMI 1640 medium, andeach 30 ml of diluted blood was layered over 20 ml Lymphoprep (Nycomed).The blood/Lymphoprep layers were centrifuged at 850 g for 20 minutes at18° C. (with no brake). Cells at the interface were removed and werewashed in RPMI 1640 by centrifugation at 850 g for 10 minutes. The cellpellets were then combined and were washed with 2×50 ml RPMI1640 at 680g for 7 minutes. Cells were resuspended to a concentration of 1×10⁶cells/ml in RPMI 1640 medium supplemented with 10% human AB serum (QuestBiomedical), L-glutamine (2 mM) and antibiotics (50 μg penicillin andstreptomycin) (Complete medium).

[0356] Test compounds were dissolved in DMSO to give 10 mM stocksolutions and were then diluted in complete medium to 20× the finalassay concentration. 10 μl of compound in solution was then added to the96-well flat bottom assay plate (final volume of 200 μl). Compounds weretested at a range of concentrations from 1×10⁻¹¹ M to 1×10⁻⁶ M. PMA andionomycin were obtained from Sigma and made up to 1 mg/ml in DMSO. Bothwere diluted to 4× the final assay concentration in complete medium. 50μl of each reagent was dispensed per well to give a final assayconcentration of 0.5 ng/ml PMA and 500 ng/ml ionomycin. Control wellsreceived 100 μl cells with PMA only (negative control 1), with ionomycinonly (negative control 2) and 100 μl cells with 50 μl PMA and 50 μlionomycin with no test compound (positive control). 100 μl completemedium was added to the negative controls. The plates were incubated at37° C. for 72 h and the cultures were pulsed with ³H-thymidine (0.5μCi/well; Amersham) for the final 6 hours. Cells were harvested on toglass fibre filter mats using a 96-well harvester (Tomtec inc., Orange,USA) and incorporated radioactivity was determined using a 1450Microbeta counter (Perkin Elmer Life Sciences, Cambridge, England). MCT1inhibitors, including Ligand 1, caused inhibition of T-cellproliferation with maximal inhibition of approximately 60%. IA₅₀ valueswere obtained from dose response curves using the 4-parameter logisticfit of a data analysis program.

[0357] IA₅₀ values are defined as the concentration of compound giving50% of the maximum possible inhibition and were obtained from doseresponse curves using the 4-parameter logistic fit of a data analysisprogram.

[0358] Experiments on Activated T-Lymphocytes

[0359] PBMCs were prepared as described above by separation overLymphoprep and T-lymphocytes were then enriched by purification on anylon wool column. Briefly, 0.6 g nylon wool was inserted into a 10 mlsyringe and this was autoclaved. The column was equilibrated with RPMI1640 containing 20% human serum (HS) for 30 min at 37° C. The PBMC wereresuspended in 1 ml of pre-warmed RPMI (20% HS) and were loaded onto thenylon wool column. The column was incubated for 45 min at 37° C.followed by elution of the T-cells by adding 10 ml RPMI (20% HS)(prewarmed to 37° C.) dropwise to the column. The T-cells obtained fromthe elution of the column were centrifuged (850 g for 5 min) andresuspended in RPMI containing L-glutamine and 5% human serum andstimulated with 0.5 ng/ml PMA and 500 ng/ml lonomycin for 48 h. T-cellswere maintained in this growth medium at an initial cell density of1×10⁶ cell per ml under standard cell culture conditions (37° C., 5%CO₂). After 48 h, the cells were harvested and prepared for the lactateand DNA synthesis assays by washing twice in RPMI 1640 and resuspendingin fresh growth medium at 1×10⁶ cells per ml.

[0360] Assay of Lactate Levels by Lactate Oxidase Enzyme Activity

[0361] a) Intracellular lactate levels: Test compounds were dissolved inDMSO to give 10 mM stock solutions and were then diluted in completemedium to 10× the final assay concentration. 100 μl of eachconcentration were added to triplicate wells of a 24-well plate. T-cellswere then added (1 ml per well) and were cultured for 4 h under standardcell culture conditions. The cells were then transferred from each wellto microfuge tubes and were centrifuged at 360 g for 5 min at 4° C. Thesupernatant was discarded and the cell pellet was resuspended in 1 ml ofice-cold PBS, pH5.0. The cells were washed twice in PBS bycentrifugation (360 g, 5 min, 4° C.) followed by resuspension of thecell pellet in 100 μl deionized water. The cells were incubated for 15min at 4° C. to allow cell lysis to occur and the cell debris wasremoved by centrifugation at 15000 g for 10 min at 4° C. 10 μl of eachsupernatant sample were used for the lactate determination.

[0362] b) Extracellular lactate levels: Test compounds were dissolved inDMSO to give 10 mM stock solutions and were then diluted in completemedium to 10× the final assay concentration. 20 μl of each concentrationwere added to triplicate wells of a 96-well plate. T-cells were thenadded (200 μl per well, 2×10⁵ cells) and were cultured for 4 h understandard cell culture conditions. The cell supernatants were collectedfrom each well after the cells were pelleted by centrifugation of theplate at 850 g for 5 min at 4° C. 10 μl of each supernatant sample wereused for the lactate determination.

[0363] Lactate Reagent (Sigma, catalogue No. 735-10) was reconstitutedaccording to the manufacturer's instructions. L-lactate (Sigma) wasprepared as a 100 mM stock solution in PBS, pH 7.5 and was diluted indistilled water to give a standard curve in the range of 12.5-200 μM.The assay was carried out in 96-well plates at room temperature. 10 μlof standard or sample were added to each well followed by 200 μl of theLactate Reagent. The plate was incubated for 15 min, and absorbance at540 nm was then determined using a SPECTRAmaxPlus spectrophotometer(Molecular Devices). The data were collected and analysed using SOFTmaxPRO software (Molecular Devices).

[0364] Assay of DNA Synthesis in Activated T-Lymphocytes

[0365] DNA synthesis was assessed by measuring the incorporation of[³H]-thymidine. The assay was carried out in a 96-well plate. 20 μl oftest compounds at 10× the final concentration were added to the platefollowed by 200 μl of T-cells per well (2×10⁵ cells). The cells werecultured for 4 h under standard cell culture conditions and cultureswere pulsed with ³H-thymidine (0.5 μCi/well; Amersham) for the finalhour of the incubation. Cells were harvested on to glass fibre filtermats using a 96-well harvester (Tomtec inc., Orange, USA) andincorporated radioactivity was determined using a 1450 Microbeta counter(Perkin Elmer Life Sciences, Cambridge, England).

[0366] Results

[0367] Compounds caused a dose-dependent decrease in the rate of [³H]thymidine incorporation and extracellular lactate concentration in 2 dayactivated T-lymphocytes. The maximum level of inhibition caused byLigand 1 was 48.6±2.1% in the assay of [³H] thymidine incorporation and30±1.9% inhibition in the assay of extracellular lactate. These valuesare representative of the maximum inhibition levels observed for thecompounds shown below.

[0368] Ligand 1 caused a dose-dependent increase in the intracellularlactate concentration from below the limit of detection (6.25 nmoles per10⁶ cells) to concentrations in the range of 26-37 nmoles per 10⁶ cells.

[0369] Mean±s.e.mean of pIA50 values (mean±range for n=2) TABLE 10Extracellular Intracellular Proliferation Ligand DNA Lactate Lactate (3days) 1 9.19 ± 0.03 9.16 ± 0.03 9.39 ± 0.07 9.46 ± 0.22 (n = 8) (n = 8)(n = 6) (n = 28) 2 8.87 ± 0.13 9.10 ± 0.17 9.26 ± 0.06 9.42 ± 0.29 (n =3) (n = 3) (n = 3) (n = 3)  5 8.57 ± 0.21 8.42 ± 0.27 8.64 ± 0.21 8.78 ±0.28 (n = 4) (n = 4) (n = 4) (n = 10)

[0370] Ligand 5:6-[(4,5-Dichloro-2-methyl-1H-imidazol-1-yl)methyl]-1,2,3,4-tetrahydro-N-methoxy-N,3-dimethyl-1-(2-methylpropyl)-2,4-dioxo-thieno[2,3-d]pyrimidine-5-carboxamideLigand 5 appears in International patent Application NumberPCT/GB02/03250

EXAMPLE 12 Inhibition of B-Lymphocyte Proliferation

[0371] Splenic B-lymphocytes were obtained from Balb/c mice bydisruption of the spleen through a nylon sieve. The resultant cellularsuspension was washed three times by centrifugation. The spleen cellswere then plated out in flat-bottomed 96 well microtitre plates at aconcentration of 2-4×10⁵ cells per well. B-lymphocyte stimulators wereadded with or without compound. Stimulators employed werelipopolysaccharide (LPS) at 50 μg/ml, 8-mercaptoguanosine (8MG) at 100μg/ml, or goat anti mouse IgM at 40 μg/ml. Ligand 2, a compound with MCTinhibitory activity, was added to the cultures in a concentration rangeof 10⁻¹⁰M to 10⁻⁵M. The cultures were incubated for 48 hours at 37° C.and pulsed with tritiated thymidine for the final 6 hours. The cellswere harvested as described previously and thymidine uptake used as ameasure of DNA synthesis.

[0372] Ligand 2 partially inhibited the proliferative response of theB-lymphocytes to LPS (IA₅₀=5×10⁻⁹M), and 8MG (IA₅₀=10⁻⁸M). Inhibition ofthe proliferative response to goat anti-mouse IgM was weak(IA₅₀=>10⁻⁷M).

EXAMPLE 13 In Vivo Activity of Compounds

[0373] Graft Versus Host Response

[0374] Compounds have been tested in the rat Graft versus Host Response(GVHR), which represents the immune elements associated with transplantrejection. The model was first described by Ford et al. (1970). Theassay consisted of injecting a 100 ul volume 5×10⁷-1×10⁹ spleen cellsfrom dark agouti (DA) rats into the right hind footpads of DA/Lewis F1hybrid rats. A similar number of DA/Lewis spleen cells were injectedinto the right hind footpads to act as controls. The DA grafted cellswere recognised by the recipient DA/Lewis rats as having “self”antigenic components whereas the DA graft cells recognised the “Le”elements of the F1 hybrids as being foreign and subsequently respondedby a proliferative response. The increase in proliferation was measuredby an increase of weight of the right lymph node compared to the controlleft lymph node. When the rats were dosed with compounds active in theMCT binding screen and the in vitro proliferation assays, thesecompounds were found to effectively inhibit the development of the GVHR.

[0375] Compounds were dosed either once or twice daily from the day ofcellular challenge in the footpads until termination 7 days later. Themost active compounds tested gave an ED50 of 1-3 mg/kg dosed by thesubcutaneous route.

[0376] Inhibition of Murine Antibody Production

[0377] Balb/c mice were immunised with 0.5 mg/kg ovalbumin (OVA) and 200mg/kg aluminium hydroxide gel in saline intraperitoneally and left for29 days before being re-challenged intraperotoneally with 0.5 mg/kgovalbumin in buffered saline vehicle and left for a further 10 days.Compound was dosed daily from re-challenge to termination prior to serumcollection. Control groups were dosed with compound vehicle only. Oncompletion of dosing, serum samples were taken from the mice andanalysed for total and specific levels of IgE.

[0378] For total IgE, microtitre plates were coated with 5 ug/mlmonoclonal rat anti-mouse IgE in phosphate buffered saline (PBS) andincubated overnight at 4° C. The plates were then washed four times withPBS containing 0.05% Tween 20 and then blocked with 1% BSA in PBS atroom temperature for 2 hours. This was followed by two further platewashings.

[0379] The serum samples and IgE standards were added to the wells induplicate and incubated overnight. The plates were then washed a furtherfour times before adding 50 ul biotinylated monoclonal rat anti-mouseIgE appropriately diluted in 0.1% BSA in PBS for 2 hours. The plate waswashed for a further four times before adding 50 ul streptavidinalkaline phosphatase conjugate appropriately diluted in 1% BSA in PBS atroom temperature for 50 minutes. The plate was washed a further fourtimes before enzyme substrate (paranitrophenyl phoshate in 1Mdiethanolamine buffer pH 9.8) at 1 mg/ml was added. Once the colourreaction developed, the plate was read at 405 nm. Levels of total IgE inserum samples were extrapolated from curve obtained with IgE standards.

[0380] For specific IgE, the methodology was similar except for thefollowing. The microtitre plates were coated with 50 ug/ml ratanti-mouse IgE. The serum samples or normal control sera were added tothe washed plates. Biotinylated OVA diluted in 0.1% BSA in PBS was usedas enzyme marker.

[0381] The activity of the MCT inhibitor Ligand 1 dosed at 3 and 30mg/kg by the subcutaneous route for 10 days following antigenic boostwas 67% and 84% inhibition respectively for total IgE and 64% and 53%inhibition respectively for OVA specific IgE.

[0382] To measure the effect of the compound on IgG2a production, Balb/cmice were immunised with ovalbumin in poly I:C adjuvant(polyinosinic:polycytidylic acid adjuvant) and left for 14 days beforebeing re-challenged with ovalbumin in buffered saline vehicle and leftfor a further 7 days. Serum samples were taken from the mice andanalysed for total and specific IgG2a. Compound was dosed daily fromre-challenge to termination prior to serum collection. For total IgG2A,microtitre plates were coated with 5 ug/ml of goat anti-mouse IgG2a inPBS and incubated overnight at 4° C. The plates were then washed fourtimes with PBS containing 0.05% Tween 20 and then blocked with 1% BSA atroom temperature for 2 hours. This was followed by two further platewashings. The serum samples and IgG2a standards were added to the wellsin duplicate and incubated at 4° C. overnight. The plates were thenwashed a further four times before adding 50 ul alkaline phosphataseconjugated goat anti-mouse IgG appropriately diluted in 0.1% BSA in PBSand left at room temperature for an hour. The plate was washed a furtherfour times before enzyme substrate (p-nitrophenyl phosphate in 1Mdiethanolamine buffer pH 9.8) at 1 mg/ml was added. Once the colourreaction developed, the absorbance of the wells at 405 nm was read in aspectrophotometer. Levels of total IgG2a in serum samples wereextrapolated from the curve obtained with IgG2a standards.

[0383] For specific IgG2a, the methodology was similar except forfollowing. The microtitre plates were coated with 50 ug/ml OVA. Theserum samples or normal control sera were added to the washed plates.Alkaline phosphatase conjugated goat anti-mouse IgG2a was used as enzymemarker.

[0384] The activity of the MCT inhibitor Ligand 1 at 3 and 30 mg/kgdosed by the subcutaneous route for 7 days following antigenic boost ontotal and specific IgG2a production gave ED50's of 30 mg/kg and 10 mg/kgrespectively.

EXAMPLE 14 Proliferation of the Human Erythroleukaemia Cell Line K562

[0385] K562 cells were cultured in RPMI1640 medium (Gibco) supplementedwith 2 mM L-glutamine and 5% foetal calf serum (FCS)(complete medium).Ligand 1 was dissolved at a concentration of 10 mM in DMSO and wasdiluted in absolute ethanol to 20× the final concentration required inthe assay. 10 μl of the diluted solutions were then added to the wellsof a 96-well microtitre plate and the ethanol was allowed to evaporate.The K562 cells were diluted to 1×10⁵ cells per ml in complete medium and100 μl was added to each well of the plate with the addition of afurther 100 μl of complete medium to give a total volume of 200 μl. Theplates were incubated for a total of 48 h at 37° C. in 5% CO₂, with theaddition of AlamarBlue (Serotec) for the last 24 h. AlamarBlue is anoxidation-reduction indicator dye that monitors metabolic activity (highlevel of reduction) as a readout of cellular proliferation. Absorbancewas measured at 600 nm (OD₆₀₀; oxidised form) and 570 nm (OD₅₇₀; reducedform) using a Spectromax spectrophotometer (Molecular Devices). Themaximum level of proliferation (OD₅₇₀-OD₆₀₀) was determined in theabsence of Ligand 1. Ligand 1 caused a significant reduction inOD₅₇₀-OD₆₀₀ with a mean pIA₅₀ of 8.7±0.2 (n=3).

EXAMPLE 15 Lactate Uptake Studies in Cells Expressing MCT Isoforms byMeasurement of Changes in Intracellular pH

[0386] 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is apH-sensitive dye that has been used by Wang et al. (Am. J. Physiol. 267(Heart Circ. Physiol. 36): H1759-69, 1994) to measure changes inintracellular pH of single cells. As lactate entry into cells isproton-linked, the addition of exogenous lactate to cells expressingfunctionally active MCT isoforms results in a significant decrease inintracellular pH. Lactate uptake by this method can be measured in cellsendogenously expressing MCT isoforms (e.g., K562 and MDA-MB231 cells) orin cells transfected with cloned MCT isoforms (e.g., MCT1, 3 and 4)expressed in SF9 or INS-1 cells as follows. The construction of plasmidscomprised of human MCT1, 2, 3, and 4 inserted into the mammalianexpression vector pCDNA3 is described in Example 3. INS-1 cells weretransfected with pcDNA3-hMCT1 using the Fugene™ 6 Transfection Reagent(Roche Molecular Biochemicals, Indianapolis, Ind., USA) to manufacturersinstructions. Stably transfected cells were selected with 100 μg/mlGenetecin (Gibco Laboratories, Grand Island, N.Y.) to generate INS-1MCT1 mixed populations, which were subsequently dilution cloned togenerate INS-1 MCT1 clones. The same procedure was performed to generatemixed populations and clones for pCDNA3-hMCT2, pCDNA3-hMCT3 andpCDNA3-hMCT4.

[0387] A 75 cm² flask of INS-1 cells (clones or mixed populations)transfected with either pCDNA3-hMCT1, pCDNA3-hMCT2, pCDNA3-hMCT3,pCDNA3-hMCT4 or pCDNA3.1 was washed with complete medium (RPMI1640containing 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 0.00035%β-mercaptoethanol) and the cells removed by incubation with 3 ml ofaccutase (Innovative Cell Technologies, La Jolla, Calif., USA.) for 5mins at 37° C. 3 ml of complete medium was added to each flask and thecells removed by gentle agitation. The cell suspension was removed andcentrifuged (465×g for 5 min). The resultant cell pellet was resuspendedin 5 ml of RPMI 1640 media without serum. Cells were counted using ahaemocytometer and resuspended in RPMI 1640 medium without serum at1.0×10⁶ cells/ml. Cells were loaded with BCECF by addition of 1 μl of 1mM stock solution per 1 ml of cells to give a final concentration of 1μM. The BCECF was incubated with the cells for 30 min at roomtemperature in the dark. Following loading with BCECF, the cellsuspensions were centrifuged (465×g for 5 min) and then washed twice inTyrodes buffer (140 mM NaCl, 4 mM KCl, 0.2 mM CaCl₂, 1 mM MgCl₂, 10 mMHEPES, 10 mM glucose, pH 7.4). The BCECF-loaded cells were thenresuspended at a concentration of 1.0×10⁷ cells/ml in Tyrodes buffer.

[0388] Following BCECF labelling, 10 μl of the relevant cell suspensionwas added to each well of a 96 well plate (Biocat Poly-D-Lysine coatedclear-bottomed black plates, Becton Dickinson) and 10 μl of inhibitor orvehicle control was added per well at 10× final concentration. 90 μl ofTyrodes or pH calibration buffer (140 mM KCl, 1 mM MgCl₂, 20 mM HEPES, 1mM EGTA, 0.01 mM Nigericin at pH values from 5.39 to 8.44) was added toeach well. Plates were incubated for 60 min at room temperature in thedark. The 96 well plate was then centrifuged at 275 g for 5 mins toensure that the cells formed a monolayer on the bottom of the plate. The96 well plate was then placed in the FLEXstation and fluorescence wasmeasured using the following wavelengths: Excitation Emission Cut offLm1 490 535 555 Lm2 440 535 555

[0389] The experiment was set up so that a reading at time zero wasmade, the lactate was added to the plate, and then readings were madeevery 3 seconds for 3 mins. L(+)-lactate (Sigma) was prepared at 3 timesthe final concentration by dilution of a 1M lactate solution in Tyrodesbuffer. 50 μl diluted lactate solution was then added to each well. Theratio of fluorescence at 490 nm/440 nm was calculated and used toprepare a pH calibration curve for wells containing the pH calibrationbuffer. The pH calibration curve was then used to determine the pH oftest wells from the 490/440 nm fluorescence ratio.

[0390] The addition of exogenous lactate caused a significant reductionin the intracellular pH of INS-1 cells expressing human MCT1, 3 or 4. Nochange in intracellular pH in response to lactate addition was observedin untransfected INS-1 cells or in INS-1 cells expressing the pCDNA3.1vector. 100 nM Ligand 2 (AR-C122982) completely abolished the decreasein intracellular pH observed on addition of exogenous lactate to INS-1cells expressing human MCT1 (n=2).

[0391] Each of the publications referenced herein is hereby incorporatedby reference in its entirety. To the extent that any definitions mayconflict, the definitions set forth herein shall prevail.

1 44 1 500 PRT Homo sapiens 1 Met Pro Pro Ala Val Gly Gly Pro Val GlyTyr Thr Pro Pro Asp Gly 1 5 10 15 Gly Trp Gly Trp Ala Val Val Ile GlyAla Phe Ile Ser Ile Gly Phe 20 25 30 Ser Tyr Ala Phe Pro Lys Ser Ile ThrVal Phe Phe Lys Glu Ile Glu 35 40 45 Gly Ile Phe His Ala Thr Thr Ser GluVal Ser Trp Ile Ser Ser Ile 50 55 60 Met Leu Ala Val Met Tyr Gly Gly GlyPro Ile Ser Ser Ile Leu Val 65 70 75 80 Asn Lys Tyr Gly Ser Arg Ile ValMet Ile Val Gly Gly Cys Leu Ser 85 90 95 Gly Cys Gly Leu Ile Ala Ala SerPhe Cys Asn Thr Val Gln Gln Leu 100 105 110 Tyr Val Cys Ile Gly Val IleGly Gly Leu Gly Leu Ala Phe Asn Leu 115 120 125 Asn Pro Ala Leu Thr MetIle Gly Lys Tyr Phe Tyr Lys Arg Arg Pro 130 135 140 Leu Ala Asn Gly LeuAla Met Ala Gly Ser Pro Val Phe Leu Cys Thr 145 150 155 160 Leu Ala ProLeu Asn Gln Val Phe Phe Gly Ile Phe Gly Trp Arg Gly 165 170 175 Ser PheLeu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Ala Gly 180 185 190 AlaLeu Met Arg Pro Ile Gly Pro Lys Pro Thr Lys Ala Gly Lys Asp 195 200 205Lys Ser Lys Ala Ser Leu Glu Lys Ala Gly Lys Ser Gly Val Lys Lys 210 215220 Asp Leu His Asp Ala Asn Thr Asp Leu Ile Gly Arg His Pro Lys Gln 225230 235 240 Glu Lys Arg Ser Val Phe Gln Thr Ile Asn Gln Phe Leu Asp LeuThr 245 250 255 Leu Phe Thr His Arg Gly Phe Leu Leu Tyr Leu Ser Gly AsnVal Ile 260 265 270 Met Phe Phe Gly Leu Phe Ala Pro Leu Val Phe Leu SerSer Tyr Gly 275 280 285 Lys Ser Gln His Tyr Ser Ser Glu Lys Ser Ala PheLeu Leu Ser Ile 290 295 300 Leu Ala Phe Val Asp Met Val Ala Arg Pro SerMet Gly Leu Val Ala 305 310 315 320 Asn Thr Lys Pro Ile Arg Pro Arg IleGln Tyr Phe Phe Ala Ala Ser 325 330 335 Val Val Ala Asn Gly Val Cys HisMet Leu Ala Pro Leu Ser Thr Thr 340 345 350 Tyr Val Gly Phe Cys Val TyrAla Gly Phe Phe Gly Phe Ala Phe Gly 355 360 365 Trp Leu Ser Ser Val LeuPhe Glu Thr Leu Met Asp Leu Val Gly Pro 370 375 380 Gln Arg Phe Ser SerAla Val Gly Leu Val Thr Ile Val Glu Cys Cys 385 390 395 400 Pro Val LeuLeu Gly Pro Pro Leu Leu Gly Arg Leu Asn Asp Met Tyr 405 410 415 Gly AspTyr Lys Tyr Thr Tyr Trp Ala Cys Gly Val Val Leu Ile Ile 420 425 430 SerGly Ile Tyr Leu Phe Ile Gly Met Gly Ile Asn Tyr Arg Leu Leu 435 440 445Ala Lys Glu Gln Lys Ala Asn Glu Gln Lys Lys Glu Ser Lys Glu Glu 450 455460 Glu Thr Ser Ile Asp Val Ala Gly Lys Pro Asn Glu Val Thr Lys Ala 465470 475 480 Ala Glu Ser Pro Asp Gln Lys Asp Thr Glu Gly Gly Pro Lys GluGlu 485 490 495 Glu Ser Pro Val 500 2 478 PRT Homo sapiens 2 Met Pro ProMet Pro Ser Ala Pro Pro Val His Pro Pro Pro Asp Gly 1 5 10 15 Gly TrpGly Trp Ile Val Val Gly Ala Ala Phe Ile Ser Ile Gly Phe 20 25 30 Ser TyrAla Phe Pro Lys Ala Val Thr Val Phe Phe Lys Glu Ile Gln 35 40 45 Gln IlePhe His Thr Thr Tyr Ser Glu Ile Ala Trp Ile Ser Ser Ile 50 55 60 Met LeuAla Val Met Tyr Ala Gly Gly Pro Val Ser Ser Val Leu Val 65 70 75 80 AsnLys Tyr Gly Ser Arg Pro Val Val Ile Ala Gly Gly Leu Leu Cys 85 90 95 CysLeu Gly Met Val Leu Ala Ser Phe Ser Ser Ser Val Val Gln Leu 100 105 110Tyr Leu Thr Met Gly Phe Ile Thr Gly Leu Gly Leu Ala Phe Asn Leu 115 120125 Gln Pro Ala Leu Thr Ile Ile Gly Lys Tyr Phe Tyr Arg Lys Arg Pro 130135 140 Met Ala Asn Gly Leu Ala Met Ala Gly Ser Pro Val Phe Leu Ser Ser145 150 155 160 Leu Ala Pro Phe Asn Gln Tyr Leu Phe Asn Thr Phe Gly TrpLys Gly 165 170 175 Ser Phe Leu Ile Leu Gly Ser Leu Leu Leu Asn Ala CysVal Ala Gly 180 185 190 Ser Leu Met Arg Pro Leu Gly Pro Asn Gln Thr ThrSer Lys Ser Lys 195 200 205 Asn Lys Thr Gly Lys Thr Glu Asp Asp Ser SerPro Lys Lys Ile Lys 210 215 220 Thr Lys Lys Ser Thr Trp Glu Lys Val AsnLys Tyr Leu Asp Phe Ser 225 230 235 240 Leu Phe Lys His Arg Gly Phe LeuIle Tyr Leu Ser Gly Asn Val Ile 245 250 255 Met Phe Leu Gly Phe Phe AlaPro Ile Ile Phe Leu Ala Pro Tyr Ala 260 265 270 Lys Asp Gln Gly Ile AspGlu Tyr Ser Ala Ala Phe Leu Leu Ser Val 275 280 285 Met Ala Phe Val AspMet Phe Ala Arg Pro Ser Val Gly Leu Ile Ala 290 295 300 Asn Ser Lys TyrIle Arg Pro Arg Ile Gln Tyr Phe Phe Ser Phe Ala 305 310 315 320 Ile MetPhe Asn Gly Val Cys His Leu Leu Cys Pro Leu Ala Gln Asp 325 330 335 TyrThr Ser Leu Val Leu Tyr Ala Val Phe Phe Gly Leu Gly Phe Gly 340 345 350Ser Val Ser Ser Val Leu Phe Glu Thr Leu Met Asp Leu Val Gly Ala 355 360365 Pro Arg Phe Ser Ser Ala Val Gly Leu Val Thr Ile Val Glu Cys Gly 370375 380 Pro Val Leu Leu Gly Pro Pro Leu Ala Gly Lys Leu Val Asp Leu Thr385 390 395 400 Gly Glu Tyr Lys Tyr Met Tyr Met Ser Cys Gly Ala Ile ValVal Ala 405 410 415 Ala Ser Val Trp Leu Leu Ile Gly Asn Ala Ile Asn TyrArg Leu Leu 420 425 430 Ala Lys Glu Arg Lys Glu Glu Asn Ala Arg Gln LysThr Arg Glu Ser 435 440 445 Glu Pro Leu Ser Lys Ser Lys His Ser Glu AspVal Asn Val Lys Val 450 455 460 Ser Asn Ala Gln Ser Val Thr Ser Glu ArgGlu Thr Asn Ile 465 470 475 3 504 PRT Homo sapiens 3 Met Gly Ala Gly GlyPro Arg Arg Gly Glu Gly Pro Pro Asp Gly Gly 1 5 10 15 Trp Gly Trp ValVal Leu Gly Ala Cys Phe Val Val Thr Gly Phe Ala 20 25 30 Tyr Gly Phe ProLys Ala Val Ser Val Phe Phe Arg Ala Leu Met Arg 35 40 45 Asp Phe Asp AlaGly Tyr Ser Asp Thr Ala Trp Val Ser Ser Ile Met 50 55 60 Leu Ala Met LeuTyr Gly Thr Gly Pro Val Ser Ser Ile Leu Val Thr 65 70 75 80 Arg Phe GlyCys Arg Pro Val Met Leu Ala Gly Gly Leu Leu Ala Ser 85 90 95 Ala Gly MetIle Leu Ala Ser Phe Ala Thr Arg Leu Leu Glu Leu Tyr 100 105 110 Leu ThrAla Gly Val Leu Thr Gly Leu Gly Leu Ala Leu Asn Phe Gln 115 120 125 ProSer Leu Ile Met Leu Gly Leu Tyr Phe Glu Arg Arg Arg Pro Leu 130 135 140Ala Asn Gly Leu Ala Ala Ala Gly Ser Pro Val Phe Leu Ser Ala Leu 145 150155 160 Ser Pro Leu Gly Gln Gln Leu Leu Glu Arg Phe Gly Trp Arg Gly Gly165 170 175 Phe Leu Leu Leu Gly Gly Leu Leu Leu His Cys Cys Ala Cys GlyAla 180 185 190 Val Met Arg Pro Pro Pro Gly Pro Gly Pro Arg Pro Arg ArgAsp Ser 195 200 205 Ala Gly Asp Arg Ala Gly Asp Ala Pro Gly Glu Ala GluAla Asp Gly 210 215 220 Ala Gly Leu Gln Leu Arg Glu Ala Ser Pro Arg ValArg Pro Arg Arg 225 230 235 240 Arg Leu Leu Asp Leu Ala Val Cys Thr AspArg Ala Phe Ala Val Tyr 245 250 255 Ala Val Thr Lys Phe Leu Met Ala LeuGly Leu Phe Val Pro Ala Ile 260 265 270 Leu Leu Val Asn Tyr Ala Lys AspAla Gly Val Pro Asp Thr Asp Ala 275 280 285 Ala Phe Leu Leu Ser Ile ValGly Phe Val Asp Ile Val Ala Arg Pro 290 295 300 Ala Cys Gly Ala Leu AlaGly Leu Ala Arg Leu Arg Pro His Val Pro 305 310 315 320 Tyr Leu Phe SerLeu Ala Leu Leu Ala Asn Gly Leu Thr Asp Leu Ser 325 330 335 Ser Ala ArgAla Arg Ser Tyr Gly Ala Leu Val Ala Phe Cys Val Ala 340 345 350 Phe GlyLeu Ser Tyr Gly Met Val Gly Ala Leu Gln Phe Glu Val Leu 355 360 365 MetAla Ala Val Gly Ala Pro Arg Phe Pro Ser Ala Leu Gly Leu Val 370 375 380Leu Leu Val Glu Ala Ala Ala Val Leu Ile Gly Pro Pro Ser Ala Gly 385 390395 400 Arg Leu Val Asp Ala Leu Lys Asn Tyr Glu Ile Ile Phe Tyr Leu Ala405 410 415 Gly Ser Glu Val Ala Leu Ala Gly Val Phe Met Ala Val Ala ThrAsn 420 425 430 Cys Cys Leu Arg Cys Ala Lys Ala Ala Pro Ser Gly Pro GlyThr Glu 435 440 445 Gly Gly Ala Ser Asp Thr Glu Asp Ala Glu Ala Glu GlyAsp Ser Glu 450 455 460 Pro Leu Pro Val Val Ala Glu Glu Pro Gly Asn LeuGlu Ala Leu Glu 465 470 475 480 Val Leu Ser Ala Arg Gly Glu Pro Thr GluPro Glu Ile Glu Ala Arg 485 490 495 Pro Arg Leu Ala Ala Glu Ser Val 5004 465 PRT Homo sapiens 4 Met Gly Gly Ala Val Val Asp Glu Gly Pro Thr GlyVal Lys Ala Pro 1 5 10 15 Asp Gly Gly Trp Gly Trp Ala Val Leu Phe GlyCys Phe Val Ile Thr 20 25 30 Gly Phe Ser Tyr Ala Phe Pro Lys Ala Val SerVal Phe Phe Lys Glu 35 40 45 Leu Ile Gln Glu Phe Gly Ile Gly Tyr Ser AspThr Ala Trp Ile Ser 50 55 60 Ser Ile Leu Leu Ala Met Leu Tyr Gly Thr GlyPro Leu Cys Ser Val 65 70 75 80 Cys Val Asn Arg Phe Gly Cys Arg Pro ValMet Leu Val Gly Gly Leu 85 90 95 Phe Ala Ser Leu Gly Met Val Ala Ala SerPhe Cys Arg Ser Ile Ile 100 105 110 Gln Val Tyr Leu Thr Thr Gly Val IleThr Gly Leu Gly Leu Ala Leu 115 120 125 Asn Phe Gln Pro Ser Leu Ile MetLeu Asn Arg Tyr Phe Ser Lys Arg 130 135 140 Arg Pro Met Ala Asn Gly LeuAla Ala Ala Gly Ser Pro Val Phe Leu 145 150 155 160 Cys Ala Leu Ser ProLeu Gly Gln Leu Leu Gln Asp Arg Tyr Gly Trp 165 170 175 Arg Gly Gly PheLeu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val 180 185 190 Cys Ala AlaLeu Met Arg Pro Leu Val Val Thr Ala Gln Pro Gly Ser 195 200 205 Gly ProPro Arg Pro Ser Arg Arg Leu Leu Asp Leu Ser Val Phe Arg 210 215 220 AspArg Gly Phe Val Leu Tyr Ala Val Ala Ala Ser Val Met Val Leu 225 230 235240 Gly Leu Phe Val Pro Pro Val Phe Val Val Ser Tyr Ala Lys Asp Leu 245250 255 Gly Val Pro Asp Thr Lys Ala Ala Phe Leu Leu Thr Ile Leu Gly Phe260 265 270 Ile Asp Ile Phe Ala Arg Pro Ala Ala Gly Phe Val Ala Gly LeuGly 275 280 285 Lys Val Arg Pro Tyr Ser Val Tyr Leu Phe Ser Phe Ser MetPhe Phe 290 295 300 Asn Gly Leu Ala Asp Leu Ala Gly Ser Thr Ala Gly AspTyr Gly Gly 305 310 315 320 Leu Val Val Phe Cys Ile Phe Phe Gly Ile SerTyr Gly Met Val Gly 325 330 335 Ala Leu Gln Phe Glu Val Leu Met Ala IleVal Gly Thr His Lys Phe 340 345 350 Ser Ser Ala Ile Gly Leu Val Leu LeuMet Glu Ala Val Ala Val Leu 355 360 365 Val Gly Pro Pro Ser Gly Gly LysLeu Leu Asp Ala Thr His Val Tyr 370 375 380 Met Tyr Val Phe Ile Leu AlaGly Ala Glu Val Leu Thr Ser Ser Leu 385 390 395 400 Ile Leu Leu Leu GlyAsn Phe Phe Cys Ile Arg Lys Lys Pro Lys Glu 405 410 415 Pro Gln Pro GluVal Ala Ala Ala Glu Glu Glu Lys Leu His Lys Pro 420 425 430 Pro Ala AspSer Gly Val Asp Leu Arg Glu Val Glu His Phe Leu Lys 435 440 445 Ala GluPro Glu Lys Asn Gly Glu Val Val His Thr Pro Glu Thr Ser 450 455 460 Val465 5 494 PRT Rattus norvegicus 5 Met Pro Pro Ala Ile Gly Gly Pro ValGly Tyr Thr Pro Pro Asp Gly 1 5 10 15 Gly Trp Gly Trp Ala Val Val ValGly Ala Phe Ile Ser Ile Gly Phe 20 25 30 Ser Tyr Ala Phe Pro Lys Ser IleThr Val Phe Phe Lys Glu Ile Glu 35 40 45 Ile Ile Phe Ser Ala Thr Thr SerGlu Val Ser Trp Ile Ser Ser Ile 50 55 60 Met Leu Ala Val Met Tyr Ala GlyGly Pro Ile Ser Ser Ile Leu Val 65 70 75 80 Asn Lys Tyr Gly Ser Arg ProVal Met Ile Ala Gly Gly Cys Leu Ser 85 90 95 Gly Cys Gly Leu Ile Ala AlaSer Phe Cys Asn Thr Val Gln Glu Leu 100 105 110 Tyr Phe Cys Ile Gly ValIle Gly Gly Leu Gly Leu Ala Phe Asn Leu 115 120 125 Asn Pro Ala Leu ThrMet Ile Gly Lys Tyr Phe Tyr Lys Lys Arg Pro 130 135 140 Leu Ala Asn GlyLeu Ala Met Ala Gly Ser Pro Val Phe Leu Ser Thr 145 150 155 160 Leu AlaPro Leu Asn Gln Ala Phe Phe Gly Ile Phe Gly Trp Arg Gly 165 170 175 SerPhe Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Ala Gly 180 185 190Ser Leu Met Arg Pro Ile Gly Pro Gln Gln Gly Lys Val Glu Lys Leu 195 200205 Lys Ser Lys Glu Ser Leu Gln Glu Ala Gly Lys Ser Asp Ala Asn Thr 210215 220 Asp Leu Ile Gly Gly Ser Pro Lys Gly Glu Lys Leu Ser Val Phe Gln225 230 235 240 Thr Val Asn Lys Phe Leu Asp Leu Ser Leu Phe Thr His ArgGly Phe 245 250 255 Leu Leu Tyr Leu Ser Gly Asn Val Val Met Phe Phe GlyLeu Phe Thr 260 265 270 Pro Leu Val Phe Leu Ser Asn Tyr Gly Lys Ser LysHis Phe Ser Ser 275 280 285 Glu Lys Ser Ala Phe Leu Leu Ser Ile Leu AlaPhe Val Asp Met Val 290 295 300 Ala Arg Pro Ser Met Gly Leu Ala Ala AsnThr Arg Trp Ile Arg Pro 305 310 315 320 Arg Val Gln Tyr Phe Phe Ala AlaSer Val Val Ala Asn Gly Val Cys 325 330 335 His Leu Leu Ala Pro Leu SerThr Thr Tyr Val Gly Phe Cys Ile Tyr 340 345 350 Ala Gly Val Phe Gly PheAla Phe Gly Trp Leu Ser Ser Val Leu Phe 355 360 365 Glu Thr Leu Met AspLeu Val Gly Pro Gln Arg Phe Ser Ser Ala Val 370 375 380 Gly Leu Val ThrIle Val Glu Cys Cys Pro Val Leu Leu Gly Pro Pro 385 390 395 400 Leu LeuGly Arg Leu Asn Asp Met Tyr Gly Asp Tyr Lys Tyr Thr Tyr 405 410 415 TrpAla Cys Gly Val Ile Leu Ile Ile Ala Gly Leu Tyr Leu Phe Ile 420 425 430Gly Met Gly Ile Asn Tyr Arg Leu Val Ala Lys Glu Gln Lys Ala Glu 435 440445 Glu Lys Lys Arg Asp Gly Lys Glu Asp Glu Thr Ser Thr Asp Val Asp 450455 460 Glu Lys Pro Lys Lys Thr Met Lys Glu Thr Gln Ser Pro Ala Pro Leu465 470 475 480 Gln Asn Ser Ser Gly Asp Pro Ala Glu Glu Glu Ser Pro Val485 490 6 29 DNA Artificial Sequence synthetically generatedoligonucleotide 6 ggatccacca tgccaccagc agttggagg 29 7 31 DNA ArtificialSequence synthetically generated oligonucleotide 7 gtcgactcag actggactttcctcctcctt g 31 8 35 DNA Artificial Sequence synthetically generatedoligonucleotide 8 tgcatgatca atgccacctg cgattggcgg gccag 35 9 30 DNAArtificial Sequence synthetically generated oligonucleotide 9 tgcagctagctcagactggg ctctcctcct 30 10 30 DNA Artificial Sequence syntheticallygenerated oligonucleotide 10 agctggatcc accatgccac caatgccaag 30 11 34DNA Artificial Sequence synthetically generated oligonucleotide 11gactctcgag ttaaatgtta gtttctcttt ctga 34 12 25 DNA Artificial Sequencesynthetically generated oligonucleotide 12 atcaggatcc aggcagcgat gggcg25 13 26 DNA Artificial Sequence synthetically generated oligonucleotide13 gacacggggc ccgtgccgta gagcat 26 14 24 DNA Artificial Sequencesynthetically generated oligonucleotide 14 cggcacgggc cccgtgtcca gcat 2415 26 DNA Artificial Sequence synthetically generated oligonucleotide 15aggcccaggc ctgtgagcac cccagc 26 16 20 DNA Artificial Sequencesynthetically generated oligonucleotide 16 gttcccggat ctgctgggtt 20 1720 DNA Artificial Sequence synthetically generated oligonucleotide 17tggagcttcc ctgggtctaa 20 18 33 DNA Artificial Sequence syntheticallygenerated oligonucleotide 18 ccctctgccg gccgcctggt ggatgcgttg aag 33 1935 DNA Artificial Sequence synthetically generated oligonucleotide 19gtcaactagt cagacaccca ggggatcaac tggag 35 20 28 DNA Artificial Sequencesynthetically generated oligonucleotide 20 tgctcacagg cctgggcctggccctcaa 28 21 26 DNA Artificial Sequence synthetically generatedoligonucleotide 21 accaggcggc cggcagaggg cggtcc 26 22 25 DNA ArtificialSequence synthetically generated oligonucleotide 22 taggaagaagcccaaagagc cacag 25 23 35 DNA Artificial Sequence syntheticallygenerated oligonucleotide 23 gacttctaga gcccagccac tcagacactt gtttc 3524 27 DNA Artificial Sequence synthetically generated oligonucleotide 24gatcggatcc atgggagggg ccgtggt 27 25 26 DNA Artificial Sequencesynthetically generated oligonucleotide 25 gtcagatatc gccactcaga cacttg26 26 16 PRT Homo sapiens 26 Cys Gln Lys Asp Thr Glu Gly Gly Pro Lys GluGlu Glu Ser Pro Val 1 5 10 15 27 17 PRT Homo sapiens 27 Cys Lys Val SerAsn Ala Gln Ser Val Thr Ser Glu Arg Glu Thr Asn 1 5 10 15 Ile 28 17 PRTHomo sapiens 28 Cys Thr Glu Pro Glu Ile Glu Ala Arg Pro Arg Leu Ala AlaGlu Ser 1 5 10 15 Val 29 17 PRT Homo sapiens 29 Cys Glu Pro Glu Lys AsnGly Glu Val Val His Thr Pro Glu Thr Ser 1 5 10 15 Val 30 9 PRT Homosapiens 30 Ala Trp Arg His Pro Gln Phe Gly Gly 1 5 31 82 DNA Homosapiens 31 ccggaccaga aagacacaga aggagggccc aaggaggagg aaagtccagtcgcttggaga 60 catccacaat ttggtggtta at 82 32 82 DNA Homo sapiens 32ctagattaac caccaaattg tggatgtctc caagcgactg gactttcctc ctccttgggc 60cctccttctg tgtctttctg gt 82 33 60 DNA Homo sapiens 33 gtgtaacctcagaaagagaa actaacattg cttggagaca tccacaattt ggtggttaat 60 34 67 DNA Homosapiens 34 ctagattaac caccaaattg tggatgtctc caagcaatgt tagtttctctttctgaggtt 60 acactct 67 35 20 DNA Artificial Sequence primer 35gccatcctgc tggtgaacta 20 36 74 DNA Artificial Sequence primer 36tagctagtct agattaacca ccaaattgtg gatgtctcca agctacagac tcggcagcca 60gcctcggcct cgcc 74 37 509 PRT Homo sapiens 37 Met Pro Pro Ala Val GlyGly Pro Val Gly Tyr Thr Pro Pro Asp Gly 1 5 10 15 Gly Trp Gly Trp AlaVal Val Ile Gly Ala Phe Ile Ser Ile Gly Phe 20 25 30 Ser Tyr Ala Phe ProLys Ser Ile Thr Val Phe Phe Lys Glu Ile Glu 35 40 45 Gly Ile Phe His AlaThr Thr Ser Glu Val Ser Trp Ile Ser Ser Ile 50 55 60 Met Leu Ala Val MetTyr Gly Gly Gly Pro Ile Ser Ser Ile Leu Val 65 70 75 80 Asn Lys Tyr GlySer Arg Ile Val Met Ile Val Gly Gly Cys Leu Ser 85 90 95 Gly Cys Gly LeuIle Ala Ala Ser Phe Cys Asn Thr Val Gln Gln Leu 100 105 110 Tyr Val CysIle Gly Val Ile Gly Gly Leu Gly Leu Ala Phe Asn Leu 115 120 125 Asn ProAla Leu Thr Met Ile Gly Lys Tyr Phe Tyr Lys Arg Arg Pro 130 135 140 LeuAla Asn Gly Leu Ala Met Ala Gly Ser Pro Val Phe Leu Cys Thr 145 150 155160 Leu Ala Pro Leu Asn Gln Val Phe Phe Gly Ile Phe Gly Trp Arg Gly 165170 175 Ser Phe Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Ala Gly180 185 190 Ala Leu Met Arg Pro Ile Gly Pro Lys Pro Thr Lys Ala Gly LysAsp 195 200 205 Lys Ser Lys Ala Ser Leu Glu Lys Ala Gly Lys Ser Gly ValLys Lys 210 215 220 Asp Leu His Asp Ala Asn Thr Asp Leu Ile Gly Arg HisPro Lys Gln 225 230 235 240 Glu Lys Arg Ser Val Phe Gln Thr Ile Asn GlnPhe Leu Asp Leu Thr 245 250 255 Leu Phe Thr His Arg Gly Phe Leu Leu TyrLeu Ser Gly Asn Val Ile 260 265 270 Met Phe Phe Gly Leu Phe Ala Pro LeuVal Phe Leu Ser Ser Tyr Gly 275 280 285 Lys Ser Gln His Tyr Ser Ser GluLys Ser Ala Phe Leu Leu Ser Ile 290 295 300 Leu Ala Phe Val Asp Met ValAla Arg Pro Ser Met Gly Leu Val Ala 305 310 315 320 Asn Thr Lys Pro IleArg Pro Arg Ile Gln Tyr Phe Phe Ala Ala Ser 325 330 335 Val Val Ala AsnGly Val Cys His Met Leu Ala Pro Leu Ser Thr Thr 340 345 350 Tyr Val GlyPhe Cys Val Tyr Ala Gly Phe Phe Gly Phe Ala Phe Gly 355 360 365 Trp LeuSer Ser Val Leu Phe Glu Thr Leu Met Asp Leu Val Gly Pro 370 375 380 GlnArg Phe Ser Ser Ala Val Gly Leu Val Thr Ile Val Glu Cys Cys 385 390 395400 Pro Val Leu Leu Gly Pro Pro Leu Leu Gly Arg Leu Asn Asp Met Tyr 405410 415 Gly Asp Tyr Lys Tyr Thr Tyr Trp Ala Cys Gly Val Val Leu Ile Ile420 425 430 Ser Gly Ile Tyr Leu Phe Ile Gly Met Gly Ile Asn Tyr Arg LeuLeu 435 440 445 Ala Lys Glu Gln Lys Ala Asn Glu Gln Lys Lys Glu Ser LysGlu Glu 450 455 460 Glu Thr Ser Ile Asp Val Ala Gly Lys Pro Asn Glu ValThr Lys Ala 465 470 475 480 Ala Glu Ser Pro Asp Gln Lys Asp Thr Glu GlyGly Pro Lys Glu Glu 485 490 495 Glu Ser Pro Val Ala Trp Arg His Pro GlnPhe Gly Gly 500 505 38 487 PRT Homo sapiens 38 Met Pro Pro Met Pro SerAla Pro Pro Val His Pro Pro Pro Asp Gly 1 5 10 15 Gly Trp Gly Trp IleVal Val Gly Ala Ala Phe Ile Ser Ile Gly Phe 20 25 30 Ser Tyr Ala Phe ProLys Ala Val Thr Val Phe Phe Lys Glu Ile Gln 35 40 45 Gln Ile Phe His ThrThr Tyr Ser Glu Ile Ala Trp Ile Ser Ser Ile 50 55 60 Met Leu Ala Val MetTyr Ala Gly Gly Pro Val Ser Ser Val Leu Val 65 70 75 80 Asn Lys Tyr GlySer Arg Pro Val Val Ile Ala Gly Gly Leu Leu Cys 85 90 95 Cys Leu Gly MetVal Leu Ala Ser Phe Ser Ser Ser Val Val Gln Leu 100 105 110 Tyr Leu ThrMet Gly Phe Ile Thr Gly Leu Gly Leu Ala Phe Asn Leu 115 120 125 Gln ProAla Leu Thr Ile Ile Gly Lys Tyr Phe Tyr Arg Lys Arg Pro 130 135 140 MetAla Asn Gly Leu Ala Met Ala Gly Ser Pro Val Phe Leu Ser Ser 145 150 155160 Leu Ala Pro Phe Asn Gln Tyr Leu Phe Asn Thr Phe Gly Trp Lys Gly 165170 175 Ser Phe Leu Ile Leu Gly Ser Leu Leu Leu Asn Ala Cys Val Ala Gly180 185 190 Ser Leu Met Arg Pro Leu Gly Pro Asn Gln Thr Thr Ser Lys SerLys 195 200 205 Asn Lys Thr Gly Lys Thr Glu Asp Asp Ser Ser Pro Lys LysIle Lys 210 215 220 Thr Lys Lys Ser Thr Trp Glu Lys Val Asn Lys Tyr LeuAsp Phe Ser 225 230 235 240 Leu Phe Lys His Arg Gly Phe Leu Ile Tyr LeuSer Gly Asn Val Ile 245 250 255 Met Phe Leu Gly Phe Phe Ala Pro Ile IlePhe Leu Ala Pro Tyr Ala 260 265 270 Lys Asp Gln Gly Ile Asp Glu Tyr SerAla Ala Phe Leu Leu Ser Val 275 280 285 Met Ala Phe Val Asp Met Phe AlaArg Pro Ser Val Gly Leu Ile Ala 290 295 300 Asn Ser Lys Tyr Ile Arg ProArg Ile Gln Tyr Phe Phe Ser Phe Ala 305 310 315 320 Ile Met Phe Asn GlyVal Cys His Leu Leu Cys Pro Leu Ala Gln Asp 325 330 335 Tyr Thr Ser LeuVal Leu Tyr Ala Val Phe Phe Gly Leu Gly Phe Gly 340 345 350 Ser Val SerSer Val Leu Phe Glu Thr Leu Met Asp Leu Val Gly Ala 355 360 365 Pro ArgPhe Ser Ser Ala Val Gly Leu Val Thr Ile Val Glu Cys Gly 370 375 380 ProVal Leu Leu Gly Pro Pro Leu Ala Gly Lys Leu Val Asp Leu Thr 385 390 395400 Gly Glu Tyr Lys Tyr Met Tyr Met Ser Cys Gly Ala Ile Val Val Ala 405410 415 Ala Ser Val Trp Leu Leu Ile Gly Asn Ala Ile Asn Tyr Arg Leu Leu420 425 430 Ala Lys Glu Arg Lys Glu Glu Asn Ala Arg Gln Lys Thr Arg GluSer 435 440 445 Glu Pro Leu Ser Lys Ser Lys His Ser Glu Asp Val Asn ValLys Val 450 455 460 Ser Asn Ala Gln Ser Val Thr Ser Glu Arg Glu Thr AsnIle Ala Trp 465 470 475 480 Arg His Pro Gln Phe Gly Gly 485 39 513 PRTHomo sapiens 39 Met Gly Ala Gly Gly Pro Arg Arg Gly Glu Gly Pro Pro AspGly Gly 1 5 10 15 Trp Gly Trp Val Val Leu Gly Ala Cys Phe Val Val ThrGly Phe Ala 20 25 30 Tyr Gly Phe Pro Lys Ala Val Ser Val Phe Phe Arg AlaLeu Met Arg 35 40 45 Asp Phe Asp Ala Gly Tyr Ser Asp Thr Ala Trp Val SerSer Ile Met 50 55 60 Leu Ala Met Leu Tyr Gly Thr Gly Pro Val Ser Ser IleLeu Val Thr 65 70 75 80 Arg Phe Gly Cys Arg Pro Val Met Leu Ala Gly GlyLeu Leu Ala Ser 85 90 95 Ala Gly Met Ile Leu Ala Ser Phe Ala Thr Arg LeuLeu Glu Leu Tyr 100 105 110 Leu Thr Ala Gly Val Leu Thr Gly Leu Gly LeuAla Leu Asn Phe Gln 115 120 125 Pro Ser Leu Ile Met Leu Gly Leu Tyr PheGlu Arg Arg Arg Pro Leu 130 135 140 Ala Asn Gly Leu Ala Ala Ala Gly SerPro Val Phe Leu Ser Ala Leu 145 150 155 160 Ser Pro Leu Gly Gln Gln LeuLeu Glu Arg Phe Gly Trp Arg Gly Gly 165 170 175 Phe Leu Leu Leu Gly GlyLeu Leu Leu His Cys Cys Ala Cys Gly Ala 180 185 190 Val Met Arg Pro ProPro Gly Pro Gly Pro Arg Pro Arg Arg Asp Ser 195 200 205 Ala Gly Asp ArgAla Gly Asp Ala Pro Gly Glu Ala Glu Ala Asp Gly 210 215 220 Ala Gly LeuGln Leu Arg Glu Ala Ser Pro Arg Val Arg Pro Arg Arg 225 230 235 240 ArgLeu Leu Asp Leu Ala Val Cys Thr Asp Arg Ala Phe Ala Val Tyr 245 250 255Ala Val Thr Lys Phe Leu Met Ala Leu Gly Leu Phe Val Pro Ala Ile 260 265270 Leu Leu Val Asn Tyr Ala Lys Asp Ala Gly Val Pro Asp Thr Asp Ala 275280 285 Ala Phe Leu Leu Ser Ile Val Gly Phe Val Asp Ile Val Ala Arg Pro290 295 300 Ala Cys Gly Ala Leu Ala Gly Leu Ala Arg Leu Arg Pro His ValPro 305 310 315 320 Tyr Leu Phe Ser Leu Ala Leu Leu Ala Asn Gly Leu ThrAsp Leu Ser 325 330 335 Ser Ala Arg Ala Arg Ser Tyr Gly Ala Leu Val AlaPhe Cys Val Ala 340 345 350 Phe Gly Leu Ser Tyr Gly Met Val Gly Ala LeuGln Phe Glu Val Leu 355 360 365 Met Ala Ala Val Gly Ala Pro Arg Phe ProSer Ala Leu Gly Leu Val 370 375 380 Leu Leu Val Glu Ala Ala Ala Val LeuIle Gly Pro Pro Ser Ala Gly 385 390 395 400 Arg Leu Val Asp Ala Leu LysAsn Tyr Glu Ile Ile Phe Tyr Leu Ala 405 410 415 Gly Ser Glu Val Ala LeuAla Gly Val Phe Met Ala Val Ala Thr Asn 420 425 430 Cys Cys Leu Arg CysAla Lys Ala Ala Pro Ser Gly Pro Gly Thr Glu 435 440 445 Gly Gly Ala SerAsp Thr Glu Asp Ala Glu Ala Glu Gly Asp Ser Glu 450 455 460 Pro Leu ProVal Val Ala Glu Glu Pro Gly Asn Leu Glu Ala Leu Glu 465 470 475 480 ValLeu Ser Ala Arg Gly Glu Pro Thr Glu Pro Glu Ile Glu Ala Arg 485 490 495Pro Arg Leu Ala Ala Glu Ser Val Ala Trp Arg His Pro Gln Phe Gly 500 505510 Gly 40 500 PRT Homo sapiens 40 Met Pro Pro Met Pro Ser Ala Pro ProVal His Pro Pro Pro Asp Gly 1 5 10 15 Gly Trp Gly Trp Ile Val Val GlyAla Ala Phe Ile Ser Ile Gly Phe 20 25 30 Ser Tyr Ala Phe Pro Lys Ala ValThr Val Phe Phe Lys Glu Ile Gln 35 40 45 Gln Ile Phe His Thr Thr Tyr SerGlu Ile Ala Trp Ile Ser Ser Ile 50 55 60 Met Leu Ala Val Met Tyr Ala GlyGly Pro Val Ser Ser Val Leu Val 65 70 75 80 Asn Lys Tyr Gly Ser Arg ProVal Val Ile Ala Gly Gly Leu Leu Cys 85 90 95 Cys Leu Gly Met Val Leu AlaSer Phe Ser Ser Ser Val Val Gln Leu 100 105 110 Tyr Leu Thr Met Gly PheIle Thr Gly Leu Gly Leu Ala Phe Asn Leu 115 120 125 Gln Pro Ala Leu ThrIle Ile Gly Lys Tyr Phe Tyr Arg Lys Arg Pro 130 135 140 Met Ala Asn GlyLeu Ala Met Ala Gly Ser Pro Val Phe Leu Ser Ser 145 150 155 160 Leu AlaPro Phe Asn Gln Tyr Leu Phe Asn Thr Phe Gly Trp Lys Gly 165 170 175 SerPhe Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Ala Gly 180 185 190Ala Leu Met Arg Pro Ile Gly Pro Lys Pro Thr Lys Ala Gly Lys Asp 195 200205 Lys Ser Lys Ala Ser Leu Glu Lys Ala Gly Lys Ser Gly Val Lys Lys 210215 220 Asp Leu His Asp Ala Asn Thr Asp Leu Ile Gly Arg His Pro Lys Gln225 230 235 240 Glu Lys Arg Ser Val Phe Gln Thr Ile Asn Gln Phe Leu AspLeu Thr 245 250 255 Leu Phe Thr His Arg Gly Phe Leu Leu Tyr Leu Ser GlyAsn Val Ile 260 265 270 Met Phe Phe Gly Leu Phe Ala Pro Leu Val Phe LeuSer Ser Tyr Gly 275 280 285 Lys Ser Gln His Tyr Ser Ser Glu Lys Ser AlaPhe Leu Leu Ser Ile 290 295 300 Leu Ala Phe Val Asp Met Val Ala Arg ProSer Met Gly Leu Val Ala 305 310 315 320 Asn Thr Lys Pro Ile Arg Pro ArgIle Gln Tyr Phe Phe Ala Ala Ser 325 330 335 Val Val Ala Asn Gly Val CysHis Met Leu Ala Pro Leu Ser Thr Thr 340 345 350 Tyr Val Gly Phe Cys ValTyr Ala Gly Phe Phe Gly Phe Ala Phe Gly 355 360 365 Trp Leu Ser Ser ValLeu Phe Glu Thr Leu Met Asp Leu Val Gly Pro 370 375 380 Gln Arg Phe SerSer Ala Val Gly Leu Val Thr Ile Val Glu Cys Cys 385 390 395 400 Pro ValLeu Leu Gly Pro Pro Leu Leu Gly Arg Leu Asn Asp Met Tyr 405 410 415 GlyAsp Tyr Lys Tyr Thr Tyr Trp Ala Cys Gly Val Val Leu Ile Ile 420 425 430Ser Gly Ile Tyr Leu Phe Ile Gly Met Gly Ile Asn Tyr Arg Leu Leu 435 440445 Ala Lys Glu Gln Lys Ala Asn Glu Gln Lys Lys Glu Ser Lys Glu Glu 450455 460 Glu Thr Ser Ile Asp Val Ala Gly Lys Pro Asn Glu Val Thr Lys Ala465 470 475 480 Ala Glu Ser Pro Asp Gln Lys Asp Thr Glu Gly Gly Pro LysGlu Glu 485 490 495 Glu Ser Pro Val 500 41 1503 DNA Homo sapiens 41atgccaccag cagttggagg tccagttgga tacacccccc cagatggagg ctggggctgg 60gcagtggtaa ttggagcttt catttccatc ggcttctctt atgcatttcc caaatcaatt 120actgtcttct tcaaagagat tgaaggtata ttccatgcca ccaccagcga agtgtcatgg 180atatcctcca taatgttggc tgtcatgtat ggtggaggtc ctatcagcag tatcctggtg 240aataaatatg gaagtcgtat agtcatgatt gttggtggct gcttgtcagg ctgtggcttg 300attgcagctt ctttctgtaa caccgtacag caactatacg tctgtattgg agtcattgga 360ggtcttgggc ttgccttcaa cttgaatcca gctctgacca tgattggcaa gtatttctac 420aagaggcgac cattggccaa cggactggcc atggcaggca gccctgtgtt cctctgtact 480ctggcccccc tcaatcaggt tttcttcggt atctttggat ggagaggaag ctttctaatt 540cttgggggct tgctactaaa ctgctgtgtt gctggagccc tcatgcgacc aatcgggccc 600aagccaacca aggcagggaa agataagtct aaagcatccc ttgagaaagc tggaaaatct 660ggtgtgaaaa aagatctgca tgatgcaaat acagatctta ttggaagaca ccctaaacaa 720gagaaacgat cagtcttcca aacaattaat cagttcctgg acttaaccct attcacccac 780agaggctttt tgctatacct ctctggaaat gtgatcatgt tttttggact ctttgcacct 840ttggtgtttc ttagtagtta tgggaagagt cagcattatt ctagtgagaa gtctgccttc 900cttctttcca ttctggcttt tgttgacatg gtagcccgac catctatggg acttgtagcc 960aacacaaagc caataagacc tcgaattcag tatttctttg cggcttccgt tgttgcaaat 1020ggagtgtgtc atatgctagc acctttatcc actacctatg ttggattctg tgtctatgcg 1080ggattctttg gatttgcctt cgggtggctc agctccgtat tgtttgaaac attgatggac 1140cttgttggac cccagaggtt ctccagcgct gtgggattgg tgaccattgt ggaatgctgt 1200cctgtcctcc tggggccacc acttttaggt cggctcaatg acatgtatgg agactacaaa 1260tacacatact gggcatgtgg cgtcgtccta attatttcag gtatctatct cttcattggc 1320atgggcatca attatcgact tttggcaaaa gaacagaaag caaacgagca gaaaaaggaa 1380agtaaagagg aagagaccag tatagatgtt gctgggaagc caaatgaagt taccaaagca 1440gcagaatctc cggaccagaa agacacagaa ggagggccca aggaggagga aagtccagtc 1500tga 1503 42 1437 DNA Homo sapiens 42 atgccaccaa tgccaagtgc cccacctgtgcatccacctc cagatggagg atggggttgg 60 attgtggttg gagcagcttt tatctccattggattttcct atgcattccc caaagctgtc 120 accgtattct tcaaagaaat tcagcaaatattccacacta cctacagtga aatagcatgg 180 atttcatcca ttatgctggc tgttatgtacgcaggaggtc ctgtaagtag tgttttggtg 240 aataaatacg gcagccggcc ggtggtgatagcaggaggct tattatgctg tcttggaatg 300 gtgttggcct cctttagtag cagcgtggtacagctgtacc tcactatggg attcattaca 360 ggtttaggtt tagccttcaa cctgcaacccgccttaacca taattggcaa atacttctat 420 aggaagcgac ccatggcaaa tggattggccatggcaggaa gtcctgtttt cttaagttca 480 ttggctcctt tcaatcagta cctttttaatacttttggct ggaaaggaag cttcctgatt 540 ttgggaagtc tacttttgaa tgcctgtgtggctggttccc tcatgagacc ccttggaccc 600 aatcaaacca cttctaagtc taaaaataagactggcaaaa cagaagatga ttcaagccca 660 aagaaaatca aaacgaagaa atcaacttgggaaaaagtta ataagtattt agatttctcc 720 ctttttaagc atagaggatt tctgatatatctgtctggaa atgtcattat gttcctaggt 780 ttttttgccc ccattatatt cttggctccatatgctaaag accaaggaat tgatgagtac 840 tcggcagctt ttctgctatc tgttatggctttcgttgata tgtttgctag gccttctgta 900 ggattaattg caaactccaa atatattcgacctcgaattc agtacttctt cagttttgca 960 atcatgttca atggagtgtg tcacctcttgtgcccactgg cacaggacta cacaagcctg 1020 gtattatatg ctgtattttt tggccttggatttgggagtg ttagcagtgt tctctttgaa 1080 actctcatgg acctcgtggg tgcaccaagattttccagtg ccgtcggact tgtcacaatt 1140 gtggagtgtg gcccagttct tcttggccctcctcttgcag gtaaattggt ggatttaact 1200 ggagaatata aatacatgta catgtcctgtggggctattg tggtagcagc aagcgtgtgg 1260 ctgctcattg gcaatgctat caactatagattgcttgcaa aggaaaggaa ggaggaaaat 1320 gcaaggcaga agaccagaga atctgaacccttgagcaaat ctaaacattc ggaagatgtt 1380 aacgtcaaag tttcaaatgc acagagtgtaacctcagaaa gagaaactaa catttaa 1437 43 1515 DNA Homo sapiens 43atgggcgctg gcggcccccg gcggggcgag ggccccccag acggcggctg gggctgggtg 60gtgctgggcg cctgctttgt ggtcaccggc ttcgcctacg gcttccccaa agccgtgagc 120gtcttcttcc gcgcgctcat gcgcgacttc gatgccggct acagcgacac ggcctgggtg 180tcctccatca tgctagccat gctctacggc acgggccccg tgtccagcat cctcgtgacc 240cgctttggct gtcgcccggt gatgctggcg ggtgggctgc tggcttccgc gggcatgatc 300ctagcttcct ttgccacgcg cctcctggag ctctacctga ccgctggggt gctcacaggc 360ctgggcctgg ccctcaactt ccagccgtcg ctcatcatgc tggggctgta cttcgagcgg 420cggcggcctc tggccaacgg gctggcggcg gcgggcagcc ccgtgttcct gtccgcgctg 480tcgccgctcg gccagcagct gctggagcgc ttcggctggc gcggcggctt cctgctgctc 540ggcgggctcc tgctgcactg ctgcgcctgc ggggctgtca tgaggccgcc gcccgggccg 600ggcccgcgac cgcgcaggga cagcgccggc gaccgcgccg gggacgctcc gggcgaggcg 660gaggctgacg gtgcggggct gcagctgcgc gaggcatccc ccagggtccg gccccgccgg 720cgcctgctgg acttggcagt gtgcaccgac cgcgccttcg ccgtgtacgc cgtcaccaag 780ttcctgatgg cgctcgggct cttcgtcccc gccatcctac tggtgaacta cgccaaggac 840gcgggcgtgc ccgacaccga cgccgccttc ctgctgtcca tcgtgggctt cgtggacatc 900gtggcgcgcc cggcgtgcgg cgccctggcg ggcctggcgc gtctgcggcc gcacgtcccg 960tatctgttca gcctggccct gctggccaat gggctcacag acctgagcag cgcacgcgcg 1020cgctcctacg gcgccctcgt cgccttctgc gtcgccttcg gcctctccta cggcatggtg 1080ggcgcgctgc agttcgaggt gctcatggcg gctgtgggcg cgccccgctt ccccagtgcg 1140ctgggcctgg tgttgctcgt ggaggccgcg gctgtgctca tcggaccgcc ctctgccggc 1200cgcctggtgg atgcgttgaa gaactatgag atcatcttct acctggccgg ctctgaggtg 1260gccctggctg gggtcttcat ggctgtcgcc accaactgct gcctgcgttg tgctaaagct 1320gccccgtcag gcccaggcac tgagggcgga gccagtgaca ctgaggacgc tgaggctgaa 1380ggggactctg agcccctgcc tgttgttgca gaggaacccg gcaacctgga ggccctggag 1440gtgctcagcg cccggggcga gcccacagaa ccagaaatag aggcgaggcc gaggctggct 1500gccgagtctg tataa 1515 44 1398 DNA Homo sapiens 44 atgggagggg ccgtggtggacgagggcccc acaggcgtca aggcccctga cggcggctgg 60 ggctgggccg tgctcttcggctgtttcgtc atcactggct tctcctacgc cttccccaag 120 gccgtcagtg tcttcttcaaggagctcata caggagtttg ggatcggcta cagcgacaca 180 gcctggatct cctccatcctgctggccatg ctctacggga caggtccgct ctgcagtgtg 240 tgcgtgaacc gctttggctgccggcccgtc atgcttgtgg ggggtctctt tgcgtcgctg 300 ggcatggtgg ctgcgtccttttgccggagc atcatccagg tctacctcac cactggggtc 360 atcacggggt tgggtttggcactcaacttc cagccctcgc tcatcatgct gaaccgctac 420 ttcagcaagc ggcgccccatggccaacggg ctggcggcag caggtagccc tgtcttcctg 480 tgtgccctga gcccgctggggcagctgctg caggaccgct acggctggcg gggcggcttc 540 ctcatcctgg gcggcctgctgctcaactgc tgcgtgtgtg ccgcactcat gaggcccctg 600 gtggtcacgg cccagccgggctcggggccg ccgcgaccct cccggcgcct gctagacctg 660 agcgtcttcc gggaccgcggctttgtgctt tacgccgtgg ccgcctcggt catggtgctg 720 gggctcttcg tcccgcccgtgttcgtggtg agctacgcca aggacctggg cgtgcccgac 780 accaaggccg ccttcctgctcaccatcctg ggcttcattg acatcttcgc gcggccggcc 840 gcgggcttcg tggcggggcttgggaaggtg cggccctact ccgtctacct cttcagcttc 900 tccatgttct tcaacggcctcgcggacctg gcgggctcta cggcgggcga ctacggcggc 960 ctcgtggtct tctgcatcttctttggcatc tcctacggca tggtgggggc cctgcagttc 1020 gaggtgctca tggccatcgtgggcacccac aagttctcca gtgccattgg cctggtgctg 1080 ctgatggagg cggtggccgtgctcgtcggg cccccttcgg gaggcaaact cctggatgcg 1140 acccacgtct acatgtacgtgttcatcctg gcgggggccg aggtgctcac ctcctccctg 1200 attttgctgc tgggcaacttcttctgcatt aggaagaagc ccaaagagcc acagcctgag 1260 gtggcggccg cggaggaggagaagctccac aagcctcctg cagactcggg ggtggacttg 1320 cgggaggtgg agcatttcctgaaggctgag cctgagaaaa acggggaggt ggttcacacc 1380 ccggaaacaa gtgtctga1398

1. A method for identifying a compound having therapeutic potential, the method comprising (a) determining whether a test compound decreases monocarboxylate transport activity; and (b) if the compound decreases said activity, identifying the compound as having therapeutic potential.
 2. The method of claim 1, wherein the compound identified as having therapeutic potential is further tested in a cellular proliferation assay.
 3. The method of claim 2, wherein the cellular proliferation assay tests whether the compound inhibits proliferation of activated T lymphocytes.
 4. The method of claim 2, wherein the cellular proliferation assay tests whether the compound inhibits proliferation of cancer cells in vitro.
 5. The method of claim 2, wherein the cellular proliferation assay tests whether the compound inhibits proliferation of cancer cells in vivo.
 6. The method of claim 1, wherein the compound identified as having therapeutic potential is further tested in an in vivo or in vitro model of inflammation.
 7. The method of claim 1, wherein the compound identified as having therapeutic potential is further tested in an in vivo or in vitro model of autoimmune disease or transplant rejection.
 8. The method of claim 1, wherein step (a) comprises determining whether the compound inhibits the activity of a monocarboxylate transport protein.
 9. The method of claim 8, wherein the protein is in a cell, a cell ghost, a cell membrane fraction, or a lipid vesicle.
 10. The method of claim 1, wherein step (a) comprises determining whether the compound reduces the level of expression of a monocarboxylate transport protein in a cell.
 11. The method of claim 1, wherein the monocarboxylate transport protein is a mammalian MCT1, 2, 3, or
 4. 12. The method of claim 1, wherein the monocarboxylate transport protein is a human MCT1, 2, 3, or
 4. 13. The method of claim 1, wherein the monocarboxylate transport protein is MCT1.
 14. The method of claim 1, wherein as a result of the determination that the compound decreases monocarboxylate transport activity, the compound is identified as having therapeutic potential in the treatment of an immune-mediated disorder or cancer.
 15. The method of claim 1, wherein the determining step comprises (i) providing a cell expressing the protein; (ii) contacting the cell with the test compound; and (iii) determining whether the test compound affects one or more of the following: monocarboxylate accumulation within the cell, monocarboxylate efflux from the cell, H+ efflux from the cell, or H+ accumulation within the cell; as an indication that the test compound inhibits the protein's monocarboxylate transport activity.
 16. The method of claim 1, wherein the determining step comprises an assay selected from the group consisting of: rapid filtration of equilibrium binding mixtures, radioimmunoassays (RIA), fluorescence resonance energy transfer assays (FRET), scintillation proximity assay (SPA), measurement of intracellular pH, and the use of labelled substrates to measure transport.
 17. A method for identifying a compound having therapeutic potential, the method comprising (a) determining whether a test compound binds to a monocarboxylate transport protein; and (b) if the compound binds to the protein, identifying the compound as having therapeutic potential.
 18. The method of claim 17, wherein following step (b), the compound's ability to inhibit the activity of a monocarboxylate transport protein is tested.
 19. The method of claim 17, wherein the determining step comprises ascertaining the binding affinity of the compound for the protein.
 20. The method of claim 17, wherein the determining step comprises a competitive binding assay, using as competitive reagent a labelled second compound that specifically binds to the protein.
 21. The method of claim 17, wherein the determining step comprises providing a cell expressing the protein, or a cell membrane preparation derived from the cell, and contacting the test compound with the cell or the preparation.
 22. The method of claim 21, wherein the cell naturally expresses the protein.
 23. The method of claim 21, wherein the protein is a recombinant protein expressed by the cell, the cell being transfected with a nucleic acid encoding the protein.
 24. The method of claim 17, wherein the monocarboxylate transport protein is a mammalian MCT1, 2, 3, or
 4. 25. The method of claim 17, wherein the monocarboxylate transport protein is a human MCT1, 2, 3, or
 4. 26. The method of claim 17, wherein the monocarboxylate transport protein is MCT1.
 27. A method for producing a therapeutic composition, the method comprising carrying out the method of claim 1 to identify a compound with therapeutic potential, and mixing the compound, or a derivative thereof, with a pharmaceutically acceptable carrier.
 28. A method for producing a therapeutic composition, the method comprising carrying out the method of claim 17 to identify a compound with therapeutic potential, and mixing the compound, or a derivative thereof, with a pharmaceutically acceptable carrier.
 29. A method of producing a therapeutic composition, the method comprising carrying out the method of claim 1 to identify a compound with therapeutic potential, and manufacturing a therapeutic composition comprising the compound in accordance with practices that ensure the sterility of the composition.
 30. A method of producing a therapeutic composition, the method comprising carrying out the method of claim 17 to identify a compound with therapeutic potential, and manufacturing a therapeutic composition comprising the compound in accordance with practices that ensure the sterility of the composition.
 31. The method of claim 29, wherein the composition is labelled either for use in a method of treating an immune-mediated condition or for use in a method of treating cancer.
 32. The method of claim 30, wherein the composition is labelled either for use in a method of treating an immune-mediated condition or for use in a method of treating cancer.
 33. A compound identified by the method of claim 1, provided that the compound is not within any of Formulae I-IX.
 34. A compound identified by the method of claim 17, provided that the compound is not within any of Formulae I-IX.
 35. A compound identified by the method of claim 1, wherein the compound is at least ten times as active against one of MCT1, 2, 3, or 4, as against any other of the four.
 36. A compound identified by the method of claim 17, wherein the compound is at least ten times as active against one of MCT1, 2, 3, or 4, as against any other of the four.
 37. A method of treating a human subject in need of treatment for a disease or condition characterised by T-cell activation or cellular proliferation, the method comprising administering to the subject a compound that inhibits cellular monocarboxylate transporter activity, other than a quinazolinedione compound or a compound of formulae I to IX.
 38. A method of treating a human subject in need of treatment for an immune-mediated disorder or cancer, the method comprising administering to the subject a compound that inhibits cellular monocarboxylate transporter activity, provided that the compound is other than a quinazolinedione compound or a compound of formulae I to IX.
 39. A method of treating a human subject in need of treatment for an immune-mediated disorder or cancer, the method comprising identifying a subject as being in need of said treatment, and administering to the subject a compound that inhibits a monocarboxylate transporter other than MCT1 or MCT2.
 40. The method of claim 39, wherein the monocarboxylate transporter is MCT3 or MCT4.
 41. A method of treating a patient suffering from or likely to suffer from an immune-mediated disorder or cancer, the method comprising (i) identifying a compound as being an inhibitor of monocarboxylate transport in a cell, and (ii) administering to the patient an effective amount of the compound.
 42. The method of claim 41, wherein the compound is a broad spectrum inhibitor capable of potently inhibiting at least two monocarboxylate transport proteins.
 43. The method of claim 41, wherein the compound is at least ten times as active against one of MCT1, 2, 3, and 4, as against any other of the four.
 44. A method for treating a patient suffering from an immune-mediated disorder or cancer, the method comprising administering to said patient an effective amount of a compound that specifically reduces expression of an MCT, the compound being selected from the group consisting of an anti-sense molecule, a ribozyme molecule, an RNAi molecule and a triple helix forming molecule.
 45. A method of inhibiting T-cell or B-cell proliferation in a human, the method comprising identifying a human in need of such inhibition, and administering to the human a compound capable of specifically inhibiting monocarboxylate transport within a T-cell or B-cell. 